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ROtg,(1dMENA TO THE GRE BIOCHEMISTRY SUBJECT TEST

U\!, Marquart
GENERAL CHEMISTRY
Nucleophile electron donors
Electrophiles electron acceptors
1
st
law of thermo energy converted, never created or destroyed
2
nd
law of thermo - universal entropy always increases
Fischer projection - 2D representation of 3D structure
Stereoisomers - same chemical bonds
Enantiomers mirror image steroisomers
Diastereomers - steroisomers that are not mirror images
L-aminos, D-ribose
left-handed - clockwise up
right-handed - counterclockwise up
symmetry - cyclic (rotation about 1 axis),
dihedral (rotation along 2 axis),
icosahedral- 20 faced (virus capsid), helical
Polymerization:
head - energy for synth on growing polymer
proteins & fatty acids
tail- energy for synth on added monomer
DNA, RNA, polysaccharides
clathrate lattice of one molecule trapping another
solvation layer- shell of water around hydrophobic molecule
Proton carriers
Rossman fold - structural motif that binds NAD/NADP cofactors
[NAD+j > [NADHj
nicotinamide adenine dinucleotide
oxidations, catabolic
[NADP+ ] < [NADPH]
nicotinamide adenine dinucleotide phosphate
reductions, anabolic
FAD/FADHz - flavin adenine dinucleotide
Electron carriers
cytochromes - rapid oxidation & reduction
heme - porphyrin ring + (held by 4 nitrogen)
iron-sulfer proteins (iron-sulfer center)
quinone (ubiquinone, coenzyme Q) -1-2 electrons
flavins - from vitamin riboflavin
flux control coefficient expresses relative contrib. of enzymes to
setting rate metabolites flow through pathway. (sum of flUX control
coefficients 1)
Biological catalysts
isozymes different forms of enzyme that catalyze same reaction
ribozymes does not contain easily ionizable functional groups
abzymes catalytic antibody
Scatchard analysis - quantifies affinity blw receptor & ligand
ATP resonance & charge on Pi contribute to high potential
phosphorylation - usually at AAs wi OH side chains (5er, Thr, Tyr)
Inhibition
Agonists - structural analogs that bind receptor & mimic effects
Antagonists anologs that bind wlo triggering normal effect
competitive inhibition - binds active site, blocking catalysis
o V
max
same, Km increases (plots cross)
un competitive inhibition - bind to E5 complex at separate site
o V
max
& Km lowered (plots parallel)
mixed inhibition - binds alternative site wi or wlo substrate
o effects both V max & Km
noncompetitive inhibition very unusual
. 0 V
max
lowered, Km same (plots cross)
Alloseteric Enzymes
heterotropic effects non-substrates shift sigmOidal plot to right
homotropic effects - substrates shift sigmoidal plot to left
equation
hyperbolic dependence of Va on [51
useful for detecting allosteric regulation
neither V
max
nor Km immediately clear
double-reciprocal plot (1/V vs 1/[5])
more accurate determination ofV
max
& Km
o y-intercept - l/Vmax
ox-intercept -l/Km
V
max
- reaction rate when all active sites saturated wi substrate
Km (Michaelis constant) - [5] at]l, V
max
K
eat
(general rate constant / turnover number) # of substrate
converted to product when saturated per unit time (V
max
I [enzyme])
KealKm - specificity constant
Q (mass-action ratio) - [P]/[Rj
1
-K,n
ENZYMES
zymogen I proenzyme - inactive enzyme precursor
Condensation Reactions
synthases - catalyze condensation wlo NTP
synthetases - catalyze condensation wi NTP
Jigases catalyze condensation of two atoms wi NTP
hydroxylase - hydroxylates substrate
oxygenaes -1-2 oxygens of O
2
incorporated into substrate
diC1xygenases - both atoms of O
2
incorporated into substrate
involved in nearly all aromatic ring cleavages
dehydrogenases / oxioreductases - catalyze redox reactions
hydrolases - hydrolytic cleavage
lipases cleave tryglycerides into glycerol & fatty acids
isomerases - interconvert stereo, structural or positional isomers
mutase - transfers functional group win single molecule
protein kinases
serine/threonine kinases - primarily pp-ate serine
tyrosine kinases - Src largest family of cytoplasmic TKs
mixed-function oxidase - incorporates O
2
into steroids, etc
Cofactors
cofactor- non-protein bound to enzyme
Mg
2
+ - complexes w/ ATP to form substrate for kinases
Zn
2
+ - often found in enzyme active site
Prosthetic groups
prosthetic group - permanently bound cofactor
flavin nucleotide NAD, etc
adenine cofactor in coenzyme A, NAD+, FAD, NADP+
Coenzymes
coenzyme - organic non-protein carries groups bin enzymes
biotin - coenzyme in carboxylation reactions (C0
2
carrier)
UDP coenzyme-like carrier of hexose groups
Kinases
PKA - protein kinase A (cAMP-dep.) - serine/threonine A-kinases
PKB protein kinase B (AKT)
PKC - protein kinase C (Ca
2
+-dep.)
- binds to cytoplasmic face of PM
- stim'd by Ca
2
+ & diacylglycerol (DAG)
- atypical PKCs - not activated by Ca
2
+ & DAG (diacylglycerol)
AA synthesis
Precursors AA Details
glutamate amination
PKG - protein kinase G (cGMP-dep.)
PKR - protein kinase R (dsRNA-dep.)
AMPK AMP-activated protein kinase
metabolic master switch regulating glucose uptake,
oxidation, biogenesis of GLUT4 and mitochondria
Vitamins \ .t
'" .1. tit uw-\.oAI i\ I">'VVS
Vitamin B6 {Pyndoxal phosphate (PLP)) ....
cofactor for aminotransferase b/w glutamate & other AAs
Vitamin B12 (cobalamin) - transfer methyl groups (methionine synth)
Fat-soluble
Vitamin A (retinal)
regulates gene expo in epithelial tiss., rod & cone response
. retinoic acid activates hox genes
Vitamin D3 (cholecalciferol) - converted to 1,25-
dihydroxycholecalciferol, regulates Ca
2
+ uptake & maintenance
Vitamin E (tocopherols) - destroys oxygen radicals
Vitamin K
essential for prothrombin production
essential for glutamate y-carboxylation
Vitamin Coenzyme Reaction
Thiamine (B
1
) Thiamine Transfer of aldehydes
pyrophosphate
Riboflavin (B
2
) FADH Redox
I Niacin NADH/NADPH Redox
Pantothenic acid Coenzyme A Transfer of acyl groups
Pyridoxine{ Bb) Pyridoxal phosphate-l{ Amino acid activation
Biotin (H/B
7
) Biotin ! Transfer of CO2
Lipoic acid Upoamide Redox, acyl activation
Folic acid Tetrahydrofolate Transfer carbon groups

Cobalamin I Transfer methyl groups
AMINO ACIDS & PROTEINS
AA characteristics
ionization
in solution, AAs act as zwitterions (dipolar ions)
isoelectric point (pI) - pH where molecule has no net charge
Enzyme/lnterm Cof
glutamate dehydrogenase PLP
(glutamate)
#glutamine reg'ed by adenylation glutamine synthetase
#proline, #arginine
oxaloacetate aspartate transamination PLP
(aspa rtate) asparagine, *threonine, *Iysine + glutamine -7 asparagine
aspa rtate + cysteine *methionine
3-phosphoglycerate #serine
#glycine serine hydroxymethyltransferase H
4
folate
(serine)
#cysteine
,,,',, + "Ifid,
I
serine + methionine ( cystathionine adoMet
alanine-- transamination by PLP
pyruvate
*valine, *Ieucine, *isoleucine valine & leucine - 2
PEP *phenylalanine
+ tyrosine chorismate - branch point phenylalanine hydroxylase
erythrose 4-phosphate *tryptophan
i
PRPP *histidine i adenine + ribose I
Cofactors
pyridoxal phosphate (PLP/Vitamin B
6
)
o aminotransferase b/w glutamate & other AAs
tetrahydrofolate (HJolate) - transfers 1 carbons
o dihydrofolate reductase -dihydrofolate-7tetrahydrofolate
S-adenosyl methionine (adoMet) - transfers methyls
AA starvation
stringent response regulation coordinated w/ [AA]
uncharged tRNA bind ribosomes
stringent factor binds ribosome
stringent factor catalyzes formation of ppGpp & pppGpp
ppGpp binds rRN\ reducing synth
9 l\l\.( ">
AA degradation
Ms converted to glucose & lipids
amino transferred to a-ketoglutarate (glutamate)
branched-chain a-keto acid dehydrogenase complex
o in extrahepatic tissues (muscle, adipose, kidney, & brain)
o unbranches leucine, isoleucine & valine
non-branched-chain amino acids degraded in liver
o aminotransferase that converts branched-chain amino
acids to a-keto acids absent in liver
Degraded AA
I
pyruvate tryptophan, threonine, alanine, serine, cysteine, glycine I
acetyl-CoA phenylalanine, tyrosine, tryptophan, isoleucine, threonine i
(leucine, lysine (only ketogerifcf - ! (ketogenic)
a -ketogluta rate
(5 carbon skeletons) proline, glutamate, glutamine
I---
(converted to 5 carbons) arginine, histidine
Fumarate phenylalanine, tyrosine
succinyl-CoA threonine, methionine,d:s01eucine, valin?)
Oxaloacetate aspartate, aspargine
AA-based non-protein biomolecules
glutamate/glutamine
nitrogen donors in variety of biosynthetic reactions
y-aminobutyrate (GABA)
glycine
porphyrins (4 porphobilinogens)
+ arginine & methionine
o phosphocreatine - energy buffer in skeletal muscle
+ glutamate & cysteine
o GSH (glutathione) - redox buffer
tyrosine
cotecholamines - dopamine, norepinephrine, epinephrine
melanin (tyrosine 3-monooxygenase (tyrosinase))
+ phenylalanine
o lignin (cell walls of plants)
tryptophan
auxin - plant growth hormone
serotonin
niacin (vitamin in NAD/NADP)
arginine - nitric oxide (NO)
histidine - histamine (decarboxylated)
Protein Types
fibrous- single secondary structure (support, shape, protection)
globular- multiple secondary structure (enzyme & regulatory)
Protein Folding
molten globule - stable, partially folded protein state
I
I
I
protein disulfide isomerase (POI) - shuffles disulfide bonds
peptide prolyl cis-trans isomerase (PPI) - converts cis & trans isomers
BiP (Binding immunoglobulin protein) binds translocated proteins
in ER lumen, preventing aggregation or misfolding
Chaperones
Hsp (heat-shock proteins)
o Hsp60 - barrel shaped-chaperonin "isolation chamber"
o Hsp70 - BiP (in ER) assists folding during synth
ER chaperone proteins - calnexin & colreticulin
heat-shock response - trigged by accum. of misfolded proteins
unfolded protein response - trigged by accum. of misfolded proteins
stims increased transcription of ER chaperones
slows protein production
Pr6tein Structure
peptide bonds - usually planar (trans rather than cis config)
Amino acid backbone
o Ca.-N - ({J (phi) rotations
o Ca.-C - i./J (psi) rotations
o C-N bond in backbone is rigid
Ramachandran plot - allowed values for ({J & i./J
Secondary structures (most common aas)
a helix (glutamate, metheonine, alanine)
o normally right-handed
B sheet (isoleucine, valine, tyrosine)
connect antiparallel B sheets (glycine, proline, & asparagine)
o 8 turn - 4 residue connection
o V turn - 3 residue connection (considerably less common)
Supersecondary structures
8-a-8 loop, a-a corner, a/B barrel (8-22 B sheets)
Targeting signals
N-terminal sequences
targets for chloroplasts, rough ER, mitochon. (usually cleaved)
Internal sequences
signal patch multiple internal AAs seqs that form 3D signal
nuclear localization signal- targets protein for nucleus
o located almost anywhere (not cleaved)
Protein Glycosylation
precursor oligosaccharide - preformed sugar (14) transferred en bloc
Dolichol-lipid molecule that holds precursor oligosaccharide
oligosaccharyl transferase - next to translocator Sec61 complex
N-/inked - connected to NHl on asparagines
complex accessible to modifications
high-man nose unmodified - held close to protein
O-Iinked - on OH (serine, threonine, hydroxylysine, hydroxyproline)
[less common]
Transmembrane proteins
hydropathy plots ID membrane-spanning helices (20-30
Ms)
most glycosylated (sugars added in ER & Golgi)
Protein regulation I modification
Modification TargetAA Change
Phosphate Ser, Thr, Tyr Proteins complexing, on/off
Methyl Lysine Histone code
,
Acetyl Lysine Histone code
,Ubiquitin Lysine mono - regulates transport,
poly - degradation
Palmityl Cysteine
=
associate w/ membrane
Protein breakdown
polyubiquitination targets proteins for degradation by proteosomes
proteasome - proteasome core contains several proteases
catalytic triad '-- serine, aspartate, & histidine
found inside active site of proteases
Protein interaction domains - binding sites of complexed
PTB - phosphotyrosine-binding
Src homology - SH2 & SH3
o SH3 - binds to proline-rich stretches of polypeptide
LIPIDS
Glycerolipids
triacylglycerols (TAGs) (3 fatty acids + glycerol)
derived from phosphatidate (diacylglycerol 3-phosphate)
o 1,2-diacylglycerol intermediate
depots of metabolic fuel (lipid droplets, bud off ER)
primary fuel during fasting (converted to ketone bodies)
proteins -last cellular material to be used as energy
offatty acids from triacylglycerols recycled
glycerophospholipids / phosphoglycerides (phosphate + glycerol +
hydrocarbon tail)
phosphatidylcholine (lecithin) - primary phospholipid
o phosphatidic acid + choline choline + ATP -)
choline-Pi + CTP -) COP-choline
o methylation of phosphatidylethanolamine (in yeast)
phophatidylserine negative charge
o serine + COP-diacylglycerol
o derived from phosphatidylethanolamine (in mammals)
phosphatidylethanolamine
o (decarboxylation of phosphatidylserine)
Ether lipids
Plasmalogens
important phospholipid in myelin & heart tissue
glycerophospholipids w/ acyl chain in place of glycerol
Sphingolipids
sphingosine instead of glycerol
sites of biological recognition (define human blood groups)
ceramide - basic structural unit of sphingolipids
o fatty acid - amide linkage - sphingosine
Types
Sphingomyelins
phosphatidylcholine + ceramide
only non-glycerol membrane phospholipid
glycosphingolipids
protect membrane
important for cell recognition/adhesion
o cerebrosides myelin (galactose reSidue)
o gangliosides - contain NANA (sialic acids) negative charged
o galactosides neutral charge?
galactolipids - predominant membrane lipid in plant cells
Fatty acid synthesis
Location cytosol of higher eukaryotes, chloroplasts in plants
Details
CO
2
(or HCO
r
) needed for first step
acetyl groups transferred across mito.memb. as citrate
NADPH used from citrate leaving mito & pentose Pi pathway
fatty acyl grows by 2C from malonate (C0
2
lossed each step)
final product palmitate (16 C) or stearate (18 C)
fatty acid synthetase complex - synth from acetyl-CoA
polypeptide w/ 6 enzyme activities + acyl carrier protein (ACP)
. acetyl-CoA + CO
2
-) malonyl-ACP + acetyl -) acetoacetyl-ACP + CO
2
acetyl-CoA carboxylase - rate-limiting step
acetyl-CoA + CO
2
-) malonyl-CoA
malonyl-CoA-ACP transferase (MT) - malonyl-CoA -) acetoacetyl-ACP
Squalene
precursor of cholesterol (6 isoprene units)
Synthesis
(1) 3 acetates -) mevalonate (6-C)
(2) mevalonate -) activated isoprenes
(3) polymerization of six S-C isoprenes to squalene
HMG-CoA reductase - rate-limiting enzyme in mevalonate pathway
Cholesterol
steroid nucleus - 4 fused rings; 3 w/6-carbons & 5-carbon
principle sterol (alcohol w/ OH at C-3) in mammals
lesser amounts in plants & fungi (not in chloroplast memb)
OH of cholesterol & steroids from O
2
decreases permeability
high concentrations in lipid raft, endocytic membranes
Cholesterol Synthesis
(4) 3 isopentenyl pyrophosphate (5-carbon) condense to 15 carbon
(S) 2 is-carbon combine to 30-carbon molecule
(6) 30 carbon cyclized to form cholesterol
Major synthesis sites - ER of liver & small intestine
Biomolecules derived from Cholesterol
vitamin D (essential control of calcium metabolism)
bile acids - aid in digestion of lipids (conjugated by glycine)
steroid hormones
glucocorticoids, mineralocorticoids, sex hormones
Fatty Acid & Cholesterol Transport
Structure
apolipoproteins, phospholipids, triacylglycerols & cholesterol
chylomicrons
carry triacyiglycerols from intestine to adipocytes for storage
VWL (very low density lipop.)
carry cholesterol esters, triacylglycerols (from liver to tissues)
LOL (low density /ipop.)
lipoprotein lipase degrades TAGs, converting VLDL to LDL
transports cholesterol throughout body
taken up by endocytosis (B-l00 apolipoprotein)
HDL(high density lipop.)
removes cholesterol from blood, carrying itto liver
Lipid degradation
glycerol -7 D-glyceraldehyde 3-phosphate -7 glycolysis
Fatty acid oxidation
generate acetyl-CoA from fatty acids
animals cannot convert fatty acids into glucose b/c degradation
thru CCA does not lead to increase in oxaloacetate
Types
a oxidation - in peroxisomes when is methylated
oxidation - in mito. matrix & perox., cyclegen 2 I\lADH,
w oxidation - in liver/kidney ER, cycle gen 1 NADH & 1 FADH2
,-
13 oxidation
epimers - sugars that differ in config around 1 carbon only
anomers - isomeric form that differ at anomeric carbon
- a-isomer - OH in axial position (down)
- - OH in equatorial position (up)
mutarotation - interconvertible thru noncyclic intermediates
reducing end - end w/ free anomeric carbon (w/o glycosidic bond)
nonreducing - sugar w/ O-glycosidic bond on anomeric carbon
aldose - carbonyl group at end of chain (forming an aldehyde group)
- hemiacetal- cyclic form of aldose
- aldonic acids - oxidation of carbonyl/anomeric carbon
- uronic acids - oxidation of C6 carbon
Breaks carbon bond carbon - 2
nd
C from carboxyl (C-
3)]
ketose - carbonyl group internal
perox. & mito. in mammals, only peroxisomes in yeast/plants
malonyl-CoA shuts down
o prevents futile cycle (simul. fatty acid synth &
oxidation)
enoyl-CoA isomerase - removes cis double bonds
(u nsatu rated)
gens 1 NADH, 1 FADH
2
, 1 acetyl-CoA per cycle
odd-number fatty acids - propionyl-CoA
-7 methylmatonyl-CoA -7 succinyl-CoA -7 CCA
(1) acyl-CoA dehydrogenase
o removes H a & C, forming trans double bond
(2) enoyl-CoA hydratase
o adds H
2
0 across double bond
(3) 3-L-hydroxyacyl-CoA dehydrogenase
o converts hydroxyl to keto
(4) acyl-CoA acetyltransferase (B-ketothiolase)
o thiol group cleavage by CoA
Carnitine shuttle
carnintine transports fatty acyls (<::14 C) thru mito. inner
memb.
fatty acyl-CoA -> fatty acyl-carnitine -> fatty acyl-CoA
Enzymes
long fatty acyl-CoA synthetase
o CoA + fatty acid -7 fatty acyl-CoA
carnitine acyltransferase I
carnitine-acylcarnitine translocase
o (acyl-carnitine/carnitine transporter)
carnitine acyltransferase "
SUGARS
Structure
anomeric carbon - carbonyl carbon in carbohydrate ring structure
enantiomers - mirror image stereoisomers
diastereoisomers - non-mirror image stereo isomers
Glycolysis
substrate product Enzyme
glucose glucose 6-phosphate
hexokinase 1
ATP ADP
2 G6P fructose 6-phosphate phosphoglucose isomerase
3
F6P fructose l,6-bisphosphate
phosphofructokinase-l
ATP ADP
- hemiketal- cyclic form of ketose
pyianoses - 6 membered carbon ring
furanoses - 5 membered carbon ring
Naming
5"-6
- 20 Carbon chain, double bonds at 8-9, 11-12, 14-15
Non-Glucose Sugars
lactose -7
glucose -7 glucose 6-phosphate
galactose -7 glucose 1-phosphate
glucose -7 glucose 6-phosphate
sucrose -7
fructose -7
fructose 6-phosphate or
glyceraldehyde 3-phosphate
man nose -7 fructose -7
fructose 6-phosphate or
glyceraldehyde 3-phosphate
trehalose -7 glucose -7 glucose 6-phosphate
dextrans - bacterial + yeast polysaccharides
GLUCOSEPATHVVAYS
Glycolysis/gluconeogenesis regulation (substrate/futile cycle)
- fructose 6-phosphate <-(FBPase 1) / (PFK-1)-> fructose 1,6
bisphosphate
fructose 2,6-bisphosphate (F2,6BP)
-
regs glycolysis & gluconeogenesis thru PFK-1
-
(activates glycolysis) & FBPase-1 (inhibits gluconeogenesis)
-
made/broken down by PFK-2/FBPase-2
0 protein w/ 2 enzymatic functions
0 regulated by glucagon & insulin controls thru cAMP
Inhibits Activates
Enzyme
Directly Indirectly Directly Indirectly
PFK-l ATP, citrate F2,6BP Pi
FBPase-l(l,6) F2,6BP AMP triose
PFK-2 triose phos phates Pi insulin
FBPase-2(2,6) F6P insulin
Inhib Stim irrev
G6P NADP/NADPH? pentose Pi
yes
"p",f:i1lA"Cj
ATP, citrate ADP/AMP, F2,6P
Yes
5

dihydroxyacetone phosphate
aldolase
glyceraldehyde 3-phosphate
5 DHAP GADP/G3P triose phosphate isomerase
6
GADP/G3P 1,3-bisphosphoglycerate
GADP/G3P dehydrogenase
. NAD+ + Pi NADH
7
1,3BPG 3-phosphoglycerate
phosphoglycerate kinase
ADP AlP
S 3PG 2-phosphoglycerate phosphoglycerate mutase
9 2PG phosphoenolpyruvate enolase
10
PEP pyruvate
ADP AlP
pyruvate ki nase
\'
Gluconeogenesis
mainly from AAs from degraded proteins
primarily in liver (smaller extent in renal cortex)
2 pyruvate -> glucose (consumes 4 ATP, 2 GTP, & 2NADH)
bypass irreversible steps (1,3, & 10)
1
st
high nrg compound
ATP, alanine, acetyl-CoA,
long-chain fatty acids
yes
glucose 6-phosphatase - removes Pi from G6P
o in ER of kidney & liver only
o compartmentalized to prevent abortion of glycolysis
Glycogenesis (glucose glycogen)
Step 1- glucose + ATP glucose 6-phosphate + ADP
Bypass 3: G6P -(glucose glucose + Pi
[glucose-1-phosphate + UTP UDP-glucose + pyrophosphate]
glycogen - al-4Iink, al-6 branches every 8-12 residues
'-"-
Step 3 - F6P + ATP fructose 1,6-bisphosphate + ADP
Bypass 2: F1,6BP -(FBPase-1H F6P + Pi
Step 10 - phosphoenolpyruvate + ADP -> pyruvate + ATP
Bypass 1: OXAc -(malate malate OXAc
allows transported out of mito.
OXAc + GTP -(PEP PEP + C02 + GDP
Blood glucose level
Lowers - insulin (Ci+ channels triggers release from pancreas)
o glucose uptake, glycolysis, synth of fatty acids
o inhibits fatty acid mobilization (glycogen & TAGs storaged)
Raises - epinephrine, glucagon, cortisol
o gluconeogenesis in liver (inhibit glycolysis)
o glycolysis in muscle
o mobilizes fatty acids from triacylglycerols in adipose
Glucose transporters
GLUT4 - adipose & muscle, sequestered in absence of insulin
GLUT2 -liver, pancreas, hypothalamus, intestine (reabsorbs glucose)
Glycogenolysis
glycogen phosphorylase - phosphorylates nonreducing sugar
until 4 sugars upstream from branch
o glycogen phosphorylase alb a-active b-Iess
o controlled by pp-tion by phosphorylase kinase
o stim AMP
o inhib: ATP, glucose (binds to phosphorylase a, b)
debranching enzyme
o transferase - trans 3 sugars prior to branch to main strand
o glucosidase - hydrolyzes branched glucose
phosphoglucomutase - G1P glucose 6-phosphate
Enzyme
glycogen synthase alb (active /inactive) "L<:'
o adds glucose to glycogen (requires 8 glucose chain) ".y
glycogenin - synth glucose primer for glycogen synthase I S-
o inhib by glycogen synthase kinase (GSK-3) [3x pp'd] rr
primed wi 1 pp by casein kinase II (CKII)
Enzyme
Inhibits Activates
Directly Indirectly Directly Indirectly
phosphoprotein insulin
phosphatase 1
protein kinase A cAMP epin.
protein kinase B insulin
phosphorylase PP-1 PKA Ca"+
kinase a/b
glycogen PP-1.
r
AlP n' - i phosphorylase peptin, epin.,
phosphorylase a/b kinase, AMP
Ca
2
+
glycogen synthase PKB insulin
kinase 3
glycogen synthase GSK-3 epin., C?' PP-1, casein
kinase 2
CITRIC ACID CYCLE (Krebs)
Pyruvate dehydrogenase complex - pyruvate CO
2
+ acetyl-CoA
3 enzymes - 5 cofactors
o (24) Pyruvate dehydrogenase -thiamine
pyrophosphate (TPP)
o (24) Dihydrolipoyl transacetylase -lipoate & CoA
o (12) Dihydrolipoyl dehydrogenase - FAD & NAD
Regulation
o Inhibited by: ATP, acetyl-CoA, NADH, fatty acids
o Stimulated by: AMP, CoA, NAD+, Ca
2
+
Citric acid cycle acetyl-CoA 3NADH + 3H+ + 2C0
2
+ FADH2 + GTP
substrate product notes
1 citrate synthase
acetyl CoA citrate
oxaloacetate + H
2
O
2 aconitase citrate isocitrate
isocitrate dehyd rogenase
isocitrate a-ketoglutarate
3
NAO+ NAOH + H+, CO
2
4 a-ketoglutarate dehydrogenase complex
a-ketoglutarate succinyl-CoA heme
NAO+ NAOH + H+, CO
2
5 succinyl-CoA synthetase
succinyl-CoA succinate
6 succinate dehydrogenase
7 fumarase
GOP
succinate
Q(FAO)
GTP
fumarate
fumarate malate f- urea cycle
____ ______ -L ____ __ ________ _
Anaplerotic reactions - cofactor - thiamine pyrophosphate
- replenish CCA intermediates
- pyruvate carboxylase - pyruvate + CO
2

o biotin - prosthetic group - COf-carrier ,-
o important to gluconeogenesis & CCA
o stim acetyl-CoA
- ketone bodies - accumulate when gluconeogenesis in liver
depletes oxaloacetate (acetyl-CoA oxidized into ketones)
o synth in liver mitochondria
o acetone, acetoacetate, D-6-hydroxybutyrate (feeds brain)
o converted back to acetyl-CoA in brain, muscles for fuel
Regulation of Citric Acid Cycle
Enzyme Inhibits Stimulates

pyruvate dehydrogenase
ATP, acetyl-CoA, AMP, CoA,
NADH, fatty acids NAD+, ci+
citrate synthase
ATP, citrate, NADH,
ADP
succinyl-CoA
isocitrate dehydrogenase ATP ADP,ci+
a-ketogluta rate
NADH, succinyl-CoA
ci+
dehydrogenase complex
Glyoxylate cycle (modified CCA in plants, microorganisms, etc.)
allows synth of sugar from fat (acetyl groups to succinate)
- step 1-3, identical to CCA (acetate -> citrate -> isocitrate)
o bypasses steps in which carbon is lost as COz
isocitrate dehydrogenase
o pp'd -> glyoxylate cycle
o depp'd -> citrate acid cycle
step 4 isocitrate -(isocitrate succinate + glyoxylate
o succinate CCA
o glyoxylate + acetyl CoA -(malate malate
CCA / gluconeogenesis
Pentose Phosphate Pathway
- used to make nucleotides & coenzymes
o (RNA, DNA, ATP, NADH, FADH
2
, & CoAl
- controlled by NADP+ availability
- NADPH produced used in anabolic reactions (fatty acid synth)
G6P + 2 NADP+ + H
2
0 -> ribose 5-phosphate + CO
2
+ NADPH + 2H+
Oxidative phase
Reactants Product I Enzyme
1 IG6P + NADP+ 6-phosphoglucon I glucose 6-phosphate
I dehydrogenase
2 6PGA + H
2
0 6-phosphoglucolactonase
3 6PG + NADP+ ribulose 5-p 6-phosphogluconate
. NADPH + CO2 dehydrogenase
4 R5P ribose 5-phosphate I phosphopentose isomerase
Non-oxidative I Acceptor Regeneration phase
6 5-carbon sugar phosphates 56-carbon phosphates
enzymes:
phosphopentose isomerase/epimerase, transketolase, transaldolase
NUCLEOTIDES & NITROGEN CYCLE
Nucleotide Precursors
- AAs, ribose 5-phosphate, CO
2
, & NH3
- N for purines from glutamine, glycine,& aspartate
- aspartate precursor of pyrimidines
ribonucleotide reductase - ribose to deoxyribose (NDPs
adenylate kinase - 2 ADP ATP + AMP
de novo pathway
- pyrimidine bases assembled prior to attachment to ribose
o carbamoyl phosphate + aspartate + ribose 5-phosphate
o 5-phosphoribosyl-l-pyrophosphate (PRPP)
initial acceptor of pyrimidine ring framework
o orotate - a pyrimidine ring framework + ribose
o UMP UTP CTP (cytidylate synthetase)
o dUMP + N
5
,N
1
o-methylene-THF dTMP
- purine bases assembled on ribose
o ring built from 5-phosphoribosylamine (committed step)
o A & G derived from inosine monophosphate (IMP)
o coenzyme: tetrahydrofolate
o IMP adenylosuccinate AMP
o IMP xanthylate (XMP) GMP
salvage pathway
recycled base + 5-phosphoribosyl-1-pyrophosphate (PRPP) NMPs
Nitrogen cycle
- bacterial fixation - N
z
NH3
o 16 ATP per N2 fixed (2 ATP per electron pair added)
o symbiotic bacteria in roots of leguminous plants
o access to large energy reservoir of plant
- soil bacteria - NH3 N0
3
-
o plants - N0
3
- NH3
- all organisms - NH3 AAs nucleotides
- denitrifying soil bacteria - N0
3
- N
z
Nitrogen transport
- glutamate - intracellular ammonia carrier
- glutamine & alanine - extracellular ammonia carrier
o (excess transported to liver Urea cycle)
Transaminases
- glutamine synthetase
o main regulatory enzyme in nitrogen metabolism
o glutamate + ATP + NH3 glutamine + ADP + phosphate
- glutaminase - glutamine glutamate + NH/
- glutamate dehydrogenase
o glutamate a-ketoglutarate + NH/
Urea cycle
- birds & reptiles excrete uric acid (uricotelic animals)
- takes place in liver mitochondria & cytosol
7
- NH4'" -7 carbamoyl phosphate -7 urea
-
1 N from NH/, 1 N from aspartate
Reactants Products Enzyme
1
2ATP+ carbamoyl phosphate carbamoyl phosphate
HCOg" + NH/ 2 ADP + Pi synthetase I (mito.)
carbamoyl
ornithine
2 phosphate + citrulline + Pi
ornithine
transcarbamylase (mito.)
3
citrulline + argininosuccinate argininosuccinate
aspartate + ATP +AMP+PP; synthetase
4 argininosuccinate arginine + fumarate argininosuccinate lyase
5 arginine + H
2
O ornithine + urea arginase
DNA & REPLICATION
Tm - temperature at which half of DNA helices melt apart
denatured DNA absorbs more light than annealed
DNA forms
- B form - right-handed, Watson-Crick structure
- A form - more compact (wider, shorter) than WC
- Z form -left-handed, longer + narrower
Hoogstein pairing - DNA that allows for triplex DNA strands
G Tetraplex - 4 strand binding between guanosine
H-DNA - mirroring leads to triple helix
palindrome - inverted DNA repeat - form hairpins or cruciform
- 2-fold symmetry across 2 strands of DNA
Supercoiling can help dislodge/unbind histones
- plectonemic interwined in simple and regular way
o extended coils
- solenoidal- tight left-handed coiling
Linking number # circular DNA strand wound about another
- (LK Tw + Wr, Tw = twist #, Tw = writhe number)
catenanes -linked DNA strands (like links in a chain)
topoisomerase
- Type I breaks one strand, retains energy to reseal (no ATP)
o DNA gyrase negatively supercoils (underwinds) DNA
- Type II - breaks both strands
phase variation - DNA is inverted causing change in gene expression
mating type locus - changeable sequence where mating type stored
mating - fusion of two haploid cells
lambda & cro repressor proteins - repress other's synth
- prophage & lytic states
Centro meres
- largely consist of short, repeated DNA sequences alpha
satellite DNA
in higher organisms
over hundreds of thousands of bps
- do not seem to contain centromere-specific DNA sequences
- defined by protein assembly rather than specific DNA seq
CpG island
- CG least frequent dinucleotide
o large % near of promoters
methylation may inhibit expression
- CG suppression -loss of Cs due to methyl + deamin (C-7T)
Genome
LINEs -long interspersed nuclear elements (>Skb)
retrotransposons that lack LTRs (long terminal repeat)
SINEs short interspersed nuclear elements 500)
Retroviral-Iike elements
DNA-only transposoh 'fossils'
SSRs (single-sequence repeats/highly repetitive sequences)
generally less than 10bp repeated up to a million times
"satellite DNA" - unusual base comp causes it to migrate as
satellite bands (separated from rest of DNA)
.Transposons (mobile genetic elements)
produce many genetic variants necessary for evolution
Complexity
insertion sequences (simple)
o seq required for transposition & transposase genes
complex transposons - contain one or more genes in addition
to those needed for transposition
Types
DNA-only transposons responsible for antibiotic resistance
o Bacteria contain mostly DNA-only (few nonretroviral
retrotransposons)
o exist only as DNA move by cut-and-paste transposition or
replicative transposition
retroviral-Iike retrotransposons
o mRNA -7 reverse transcriptase -7 RNA/DNA -7 DNA/DNA
-7 integrated by intergrase
o require RNA polymerase & reverse transcriptase
nonretroviral retrotransposons
o responsible for repeated DNA seq
o require endonuclease + reverse transcriptase complex
movement
o direct transposition - movement from one site to another
o replicative transposition - replication into new site
Transposon-related terms
transposase protein that promotes transpositions
intergrase - viral transposase
cointegrate donor & target DNA intermediate during replicative
transposition
Genetic Variation
SNPs (single-nucleotide polymorphisms)
extensively characterized gene variations
Genes
haplotype blocks - sets of alleles that are inherited in clusters
complex traits - do not follow simple inheritance (often polygenic)
site-directed mutagenesis - intential mutation to determine function
transgenes - foreign or modified genes
synteny- same genes in the same order across species
pseudogene - duplicated gene deactivated by mutations
X-inactivation - occurs after Nl000 cells formed (mosaic)
x-inactivation center - XIST RNA
10% of genes remain active
unique histone composition/modification pattern
Barr body - condensed inactivated x-chromosome
dosage compensation
o inhibition, up regulation, down regulation
Chromatin
heterochromatin - highly condensed, inactive
euchromatin less condensed, active (acetylated histones) ,
o hypersensitive sites
actively transcribed regions sensitive to DNase I
AT sequences more compressible
position effect - gene silencing of positions win or nearby
heterochromatin
chromatin jiber - 30nm, zigzag model of packing
Chromosome morphology
metacentric - middle centromere
submetacentric - slightly off-center centromere
acrocentric - more off-center
telocentric centromere at or near end
isochromosome -lost arm replaced by duplicate of other
DNA-associated proteins
Histone
highly conserved, rich in lysine & arginine (+)
structure: fold - 3 helices + 2 loops
N-term tail covalent modification determines chromatin
structure
o Acetylation/methylation of Iysines
o Phosphorylation of serines
o H4 tail- important for nucleosome-nucleosome linking
C-term tail- mono or polyubiquitinated
variant core histones
o expressed at different times during development
o CENP-A - centromere-specific H3 histone
Hl-linker histone (larger & less conserved)
insulator/barrier DNA sequences - protect spread of histone code
HS4 sequence - protects l3-globin expression
Histone synthesis
20 sets of histone genes
synth mainly in S phase (1'transcription, -J.,degradation)
Histone-associated proteins
HATs (histone acetyltransjerases)
o neutralize (+) charge, destabilizing chromatin
o (type A - nuclear, type B - cytosolic)
HDACs (histone deacetylases)
o compacts chromatin, attracts other proteins
histone chaperones chromatin assembly factors
SW1/SNF & NURF - remodel chromatin
SMC proteins structural maintenance of chromosomes
Histone code
different combinations of tail modification important to
o marking
replicated chromatin
DNA damage
o Inactivation
Heterochromatin formation
Hox silencing (developmental gene silencing)
X chromosome inactivation
Code-reader complex - adapter that interprets histone code &
recruits protein complexes
nucleosome
basic unit of chromosomal organization, N200 bps
o linker DNA - up to 80bp
o nuclesome core
histone octamer (2x H2A, H2B, H3, & H4)
dimers (H3+H4, H2A+H2B) tetramers (2x H3+H4)
147bp DNA (1.65 turns), left-hand coiled
location - depends on seq, presence of other bound proteins
nucleosome sliding - movement along DNA catalyzed by
remodeling complexes
polytene chromosome
bands (dark) - inactive
interbands (light) - active
puffs (Balbiani rings) - transcriptionally active DNA
DNA binding motifs
helix-tum-helix
o regulatory proteins -lac, trp, CAP
o homeodomain - developmental
zincjingers hold together by zinc
o steroid receptors
leucine zipper motif- 2 alpha helixes - dimer
o transcriptional activators
helix-loop-helix (HLH) short a-helix + loop + long a-helix
P-Ioop
Damage/Mutation
Damage
deamination of bases (loss of amine)
o C --7 U - 100 per day per cell
loss of bases - bases dissociate from ribose (N-glycosyllinkage}
o depurination 5000 per cell per day
o depyrimidation - 500 per cell per day
UV induced pyrimidine dimers
o result in deletion or substitution of 1+ base pairs
oxidative damage occurs to double bonds & C-C bonds
hydrolytic attack - N-C bonds, phosphate
uncontrolled methylation - nitrogen, flipping out
radiation - breaks purine rings & phosphodiester bonds
Tautomeric shifts
cause G-T & A-C binding
G & T - keto --7 enol
A & C- amino --7 imino
Mutation rate
1 in 10
9
nucleotides
20,000 DNA alterations per cell per day
Mutations
point (single site} - single nucleotide or very small part
o transition one purine (or pyrimidine) to other
o transversion purine to pyrimidine & vice versa
o nonsense mutation - results in premature termination
o missense mutation different translated M
insertion / deletion loss of or gain of nucleotide(s)
o jrameshift insertion/deletion altersing reading frame
inversion - segment is reversed
translocation - segment migrates from one chromo to another
germ-line - allows mutation to be passed to progeny
polar- mutation that affects subsequent genes (polycistronic)
Gene Mutations
conditional phenotypic change under cert. conditions (temp)
loss offunction reduced gene activity (usually recessive)
o null complete loss of gene activity
gain of function - increased or inappropriate activity (usually
dominant)
o dominant-negative - mutant interferes wI normal gene
suppressor- counteracts phenotypic effect of prior mutation
o back mutation reversion to previous sequence ' '
o intragenic - within 1 gene
o extragenic - within 2 genes
Direct chemical repair
DNA photolyases - use light energy to reverse dimmers
Mismatch repair
strand-directed - depends on methylation of A resides (in GATC seq)
Excision repair
base excision repair
DNA glycosylase (exinuclease)
o removes base from DNA lesions (creates AP site)
AP endonuclease
o cuts phosphodiester at AP (apurine/apyrimidine site)
Nucleotide excision repair
cleaves phosphodiester backbone on both sides of distortion
DNA around site of damage is excised & resynthesized
TFIIH - opens up excised DNA
transcription-coupled repair
repair more efficient wIn actively transcribed genes
RNA polymerase stalls on lesions & directs repair machinery
Recombination
nonhomologous end-joining ends ligated wI loss of 1+ nucleotides
homologous recombination requires long homologous segments
exonuclease 5' end, strand invasion, branch migration
genetic exchange between DNA wI extensive homology
occurs wI highest frequency during meiosis prophase I
"resolution" by crossover or non-crossover (gene conversion)
can repair DNA damage & restart replication forks (primosome)
regulated
too much (loss of heterozygosity)
too little (mutations lead to cancer)
recombinase - integrates & excises during recombination
conservative site-specific
can result in inversion, turning genes on or off
Holliday intermediate - X form
synaptonemal complex
ladderlike structure blw homo. chromosomes during meiosis
interconnected by transverse filaments (TFs)
chiasmata - individual crossover events blw nonsister chromatids
only visible during diplotene
crossover regulation ensures at least one crossover blw homologs
crossover interference crossover inhibits formation of others
Ori (Origin of Replication)
rich in AT (weaker hydrogen bonding)
binding site for aRC, aRC recruitment proteins
10,000 in humans (excess ensures replication prior to division)
o 30,000-250,000 np bIn replication origins in mammals
replication unit - origin cluster activated together (20-80)
o activate in reproducible order
ARSs (autonomously replicating sequence)
o Euk. chromosomal origin of replication
ORC (Origin Recognition Complex)
remains associated wI origin through cell cycle
pre-RC (pre replication complex)
aRC, DNA, Cdc6 & Cdtl- helicase loading proteins
form only during Gl phase (Cdk activity low)
2x Cdc6 + 2x Cdtl- recruit 2x Mcm - helicase (6 subunits)
geminin - binds & inhibits Cdtl
disassembled during 5 phase (1'Cdk, Mcm pp'd starts repl.)
l' 5 & M-Cdk & .J, APC/C activity blocks pre-RC formation
primosome DnaB helicase + DnaG primase (lagging strand)
replisome - entire replication complex at replication fork
DNA replication
In prokaryotes:
DnaA binds 4x 9-bp
HU denatures DNA in 3x 13-bp repeats
helicase further unwinds DNA
SSB proteins bind (RPA in eukaryotes)
E. coli DNA polymerases
DNA polymerase I
removes RNA primers
Klenow fragment DNA poll wI removed 5'->3' exonuclease
DNA polymerase III
more processive than DNA poll blc of subunits
a subunit - polymerization activity
subunits - DNA clamp, prevents dissociation
E subunit - 3' to 5' exonuclease activity
T subunits -links 2 core polymerases & coordinate synthesis
Mammalian DNA polymerases
DNA polymerase a - synth primers & start of Okazaki fragments
DNA polymerase - base excision repair
DNA polymerase 6 - primary pol for both strands
DNA polymerase V - mitochondrial DNA
Viral DNA polymerases
DNA viral genomes encode own DNA polymerase
Mitochondrial & plastid DNA - circular, 2 ori, multiple copies (2-10)
RNA & TRANSCRPTION
transcriptome - all transcribed genes in an organism (mRNA + rRNA)
RNAs
snRNAs (small nuclear) splice pre-mRNA
snoRNAs (small nucleolar) - modify rRNAs
many encoded in introns of ribosomal protein genes
scaRNAs (small cajal) -modify snRNAs + snoRNAs
miRNAs & stRNAs (micro & small temporal)
complementary to 3' UTR of mRNAs
block translation & target mRNA for degradation
10
initiates RISC (RNA-induced silencing complex) formation
siRNAs - small interfering compact chromatin & degrade mRNA
others - telomere synthesis,x-chromosome inactivation
rRNA & ribosomal proteins
200 rRNA gene copies per haploid genome in humans
rRNA precursor
methylations (2'-OH positions)
isomerizations (uridine -> pseudouridine)
modified & cleaved by snoRNAs
r-protein operon
\'
also contain genes for DNA primase, RNA polymerase, EFs
r-protein in each operon functions as repressor
o repression if r-proteins exceeds rRNA
RNase 1/1- trims pre-rRNA
tRNAs
heavily modified (over 50 types of modification)
some eukaryotic tRNAs have introns
Structure
o amino acid arm
CCA 3' (added by tRNA nucleotidyl transferase),
AA attached to either 2' or 3' hydroxyl
RNase P - cleaves 5' of tRNA
RNase D - trims 3' of tRNA
o anticodon arm
o D arm & TtjJC arm
isoacceptor tRNA - different anti-codon tRNA coding for same AA
aminoacyl-tRNA synthetases Mg2+ dependent, 2 steps, ATP
wobble - inosine/inosinate in 3' position base-pairs w/ U, C, or A
5' wobble - GUG may initiate translation in prok.
o UUG & CUG even less frequent at start codon in prok.
mRNA
moncistronic - code for single polypeptide (most all euks)
polycistronic - code for multiple polypeptides (prok. & viruses)
3' UTR (untranslated region)
region bIn stop codon & poly-A tail
plays role in mRNA localization & transcription controls
repressors disrupt comm. bIn tail & cap - slowing translation
carry sites for proteins that accelerate/impede degradation
RNA editing
Prokaryotes
uracil insertion/deletion
o {gRNA (guide RNA) templates} - frameshifting
Eukaryotes
A -7 I (inosine) occurs to dsRNA
o ADARs ADR1 liver, ADR2 - brain, ADR3 - unknown
C-7U
polynucleotide phosphorylase
forms RNA-like polymers from RDPs w/o template
Involved in mRNA processing & degradation
RNA Degradation
exosome large protein complex that degrades RNA w/ exonucleases
P bodies (processing bodies) - cytosolic site of most RNA degradation
RNA Polymerase ,/ (Iv.
does not require DNA helicase (ATP)
deacetylates histone tails
limited proofreading (no nuclease activity)
requires template, nucleotides, Mg2+ & Zn
2
+
pro:
o RNA polymerase holoenzyme
core enzyme
(J subunit/factor
specificity factor
binds to promoter
dissociates from core ns into translation
eu: (3 pols - differential sensitivities to a-amanitin)
o RNA polymerase 1- 5.85, 185, 285 rRNA (45S precursor)
o RNA polymerase II - m RNA
continues trans, but overshoot lacks cap (degraded)
o RNA polymerase 111- tRNA, 55 rRNA, snRNA, other
CTD (C-terminal Domain)
o responsible for initiation & bonding of related complexes
o cap synthesizing complex
synth 5' cap (5' to 5' 7-methylguanosine - protects
eu-mRNA & aids in translation)
o cap binding complex
secures mRNA to RNA polymerase following capping
Initiation
PIC (preinitiation complex) - denatures DNA & helps position RNA
polymerase II
no uniform order of assembly
o (TFIID -7 TFIIB -7 TFIIF -7 TFIIE -7 TFIIH)
consists of 6 general transcription factors
o TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
(1) (TATA-binding protein) binds TATA box
(2) TFIIB - binds TBP & DNA
(3) TFIIA - stabilizes TFIIB-TBP complex (not necessary)
important for nonconsensus promoters which bind TBP weakly
(4) TFIIF + Pol II bind TFIIB-TBP complex
(5) TFIIE & TFIIH bind - create "closed complex"
Transcription initiation complex - polymerase + transcription factors
TFlIH - unwinds DNA to start transcription (ATP)
o Phosphorylates RNA polymerase CTD (C-terminal domain)
o Releases polymerase from promoter
Elongation
TFIIE & TFIIH - released in initial 60-70 nucleotides
Transcriptional elongation factors
suppress pausing, decrease likelihood of dissociation
coordinate activites between protein complexes
involved in posttranscriptional processing
dephosphorylated upon termination
p-TEFb (positive transcription elongation factor b) pp's CTD
Polyadenylation
upstream AAUAAA site
CstF (cleavage stim factor) & CPSF (cleavage & polyadenylation
specificity factor) transferred from tail to 3' end
PAP (polyadenylate polymerase) - adds A (from ATP)
o slow addition at first followed by rapid addition of 200-250
tail acts as timer (mRNA half-life)
o short for bacteria (seconds)
II
o varies for euks -long for B-globin (10h), short for growth
factors & regulatory proteins
tail can be extended, extending mRNA life
Poly-A binding proteins - determine final length, help direct
translation
Post-transcriptional controls of human genes)
transcription attenuation - premature termination
o used by many AA synthesis operons
riboswitch - secondary structure of mRNA that turns on/off
additional transcription
alternative RNA splicing
mRNA quality control
in cytosol
5'cap & poly-A tail recognition
EJC (exon junction complex) - stimulates translation
Nonsense-mediated mRNA decay
o stop codon in wrong place causes degradation
Exons - exon size much more uniform than intron size
Splicing & Introns
Splicesome
Composition: snRNP (RNA + protein) - snRNAs
Location: bound to pp'd tail of RNA polymerase II
SR proteins - mark junctions - recruit snRNPs
U1- 5' splice junction
BBP & U2AF - branch point, mark upstream boundary
U2 - branch point
U4/U6*U5 - triple snRNP - splices
U12-type splicesome - +10 to all U#s, recognizes diff nucleotide seq.
Self-splicing RNA - rare - phage, mitochondria, chloroplast genes
Group 1- G attacks, no lariat (guanosine cofactor)
Group II - A attacks, lariat
Trans-splicing (rare) - mRNA from diff mRNA spliced together
single 5' exon spliced onto different 3' exons
Homing - search for identical site for intron insertion
homing endonuclease copies intron into new homo. gene
retrohoming - DNA "reverse spliced" - spliced intron inserted
by endonuclease I reverse transcriptase
Transcription Regulation
Cis-elements
gene control region - gene promoter & regulatory sequences
operon
polycistronic region translated together from single promoter
gene + promoter, operator, enhancer, etc
regulon - network of operons wi common regulator
promoter
HREs (hormone response elements)
o reg. DNA seq binds hormone-receptor complex
operator - bound by repressors & activators
TATA box (Hogness) - '25 bp (bound by TBP)
Pribnow box - -lD bp (binds RNA polymerase subunit)
enhancers
additional reg. seq. in higher euks bound by activators
can enhance transcription of upstream & downstream genes
UASs (upstream activator sequences) in yeasts
UP element (upstream promoter) -40-60 bp
CAAT box - 75-80 bp
initiator - start of transcription - +1 bp
terminator - sequence that ends transcription
Trans-elements
DNA-binding transactivators - bind enhancers/UASs
effector - regulates repressor binding
inducers - prevents binding of repressor protein to DNA
gratuitous inducer- inducer cannot be metabolized
basal transcription factors - required at every RNA pol II promoter
coactivators/ corepressors - do not themselves bind on DNA
mediator - mediates activator & transcription machinery
. RNA sequences
Kozak sequence - translational start site for ribosome
leaCler - 5' end of mRNA preceding initiation codon of first gene
attenuator -lies within leader responsible for attentuation
start codon - AUG
stop codons - GAA, UGA, UAG
Specific examples
catabolite activator protein (CAP) regs certain prokaryotic genes
complexes wi cAMP, binds promoters
lac operon - transcribed wi low glucose conc. ([cAMP] high) & high
lactose conc. (Lac repressor unbound)
CREB & CREBB (cAMP response element-binding)
TRANSLATION
Ribosome
small subunit - framework for matching
o pro: 165 rRNA from 305 pre-rRNA
o eu: 185 rRNA from 455 pre-rRNA
large subunit
o pro: 235 & 55 rRNAs from 305 pre-rRNA
305 contains tRNA as "introns"
o eu: 5.85 & 285 rRNAs from 455 pre-rRNA
o catalyzes peptide bond - peptidyl transferase
o Sites:
E - exit
P - peptidyl-tRNA
A - aminoacyl-tRNA
polyribosomes/polysomes - multiple ribosomes translationally
complexed on ER surface
Initiation
Initiation in pro
Translations begins while transcription is still in progress
Initiation complex
o 305, mRNA, fMet_tRNA
fMet
, (IF-1, IF-2, IF-3), GTP, Mg2+
305 binds IF-1 + IF-3
o IF-3 prevent premature 305 + 50S binding
mRNA binds 30S-IF-1-IF-3 complex (SD seq pairs wi 165 rRNA)
fMet_tRNA
fMet
+ IF2-GTP bind AUG
50S subunit binds - GTP hydrolyzed, initiation factors depart
Initiation in Eus
elFs (eukaryotic initiation factors)
5' & 3' ends tied together - PAB (Poly(A) binding protein)
elF4F complex (eIF4E, elF4G, eIF4A) bindsto 5' cap
o [eIF4A- RNA helicase]
elF4G - binds elF4E & PAB
12
elF4F complex - associates w/ elF3 & 405 ribosomal subunit
Elongation
elongation factors (EFs) increase speed & accuracy
binding aminoacyl tRNA (aa-tRNA+EF-Tu-GTP)
o EF-Tu - mediates aminoacyl tRNA entry to ribosome
peptide bond formation (235 rRNA - peptidyl transferase)
translocation (EF-G(translocasel + GTPl [EF-G catalyzes
translocation of tRNA & mRNAJ
o large subunit translocation, then small subunit
translocation
EF-Ts - serves as GEF (guanine nucleotide exchange factor) for EF-Tu
Translocation
- initiation on free ribosomes
- binding of 5RP (sig rec. particle) to polypeptide (GTP-used)
- binding of 5RP, polypeptide, & ribosome to 5RP receptor
- dissociation of 5RP from its receptor
- translocation across membrane of ER
- cleavage of signal sequence
translocon -takes signal seq across ER memb
Termination
- termination codon in the A site
Posttranslational modifications
In Lumen of ER and/or Golgi
Cutting
N-term & C-term modifications removed or modified
Loss of signal sequence
proteolytic processing - inactive to active
Modification
Phosphorylation 5er/Thr/Tyr
Carboxylation ..... Glu
Addition
Glycosylation usually occurs in Golgi
o Asn - N-Iinked oligosaccharides
o 5er/Thr - O-linked oligosaccharides
o isoprenyl groups - Cys - thioether bond
(helps anchor protein in mem.)
o prosthetic groups - biotin, heme
Folding
disulfide cross-links
quaternary structure - complexing of subunits
Translational regulation
phosphorylation of initiation factors
RNAi - miRNAs, stRNAs bind mRNA (to 3' UTR)
o prevent translation initiation
disrupt elF4E & elF4G interaction
mRNA
stop codons bind release factor UAA, UAG, UGA
causes binding of water molecule instead of amino acid
RNA interference
dsRNA inhibits genes w/ complementary sequence
can lead to heterchromatin formation
dsRNA -7 2'-S'oligo(A)synthetase -7 RNase L (destroys aU RNA)
dsRNA -7 Protein kinase R -7 apoptosis
high energy phosphate bonds split for each new peptide bond
2 consumed in charging of tRNA w/ AA (ATP -> AMP)
2 during synthesis in ribosome
extra energy consumed w/ mistaken connection
Regulation & modification
Translational repressors bind 3' UTR (untranslated region) of mRNA
block interaction w/ initiation factors or ribosome
prevent or slow translation
Reading Frame
ORF (open-reading frame) lacking termination win codons
translation recoding
proceeding seq. causes codon to be translated differently
translational frameshifting
shift of reading frame as ribosome is translating mRNA
.leaky scanning - ignoring of AUG codon, produces different proteins
" different or no N-terminal signal sequence
IRES (internal ribosome entry site) - bypass need for 5' cap & elF4E
CELLULAR COMPONENTS
Nucleus
Subnuclear structures
Cajal bodies & GEMS (Gemini of Cajal bodies) - srioRNAs &
snRNAs modify al!lItil8g!!!l11fjli i . INS
IGCs (lnterchromatin granule clusters) - stockpiled mature
snRNPs
nucleolus - site ofrRNA transcription
o NOR (nucleolar organizing region) - region containing 455
pre-rRNA genes around which nucleolus forms
Nuclear lamina
protein meshwork, provides support for nuclear membrane
disassembles through phosphorylation by Cdk
Nuclear pore complex (NPCs)
aqueous, permeable to small molecules 50,000 daltons)
octagonal w/ long filaments
Karyopherins (nuclear transport receptors)
o Export
5' cap first CBC (cap-binding complex)
sheds some proteins (nuclear-restricted proteins)
eBe & nuclear export receptor in cytosQL.;
Ran-GEF in nucleus causes
o Import erf' .) ({".
Ran-GAP in cytosol causes
importin bound (via FG-repeats)
Nuclear import receptors - bind NPC signal
Endoplasmic Reticulum
lumen/cisternal space
cytochrome P450 system
o oxidative metabolism in ER of liver & small intestine
o drug metabolism & detoxification of foreign substances
sequester ci+ - specific regions of ER specialized for storage
lipid synth - most lipids synth in cytosolic leaf of ER
membrane
o cholesterol, ceramide & fatty acid
protein modification
o glycosylation
o proetolytic processing - in ER & post-Golgi
Transport into ER
Most proteins imported during translation
Signal-recognition particle (SRP) - cycles bin ER & cytosol
Sec61 complex - tetra mer, 1 pore + bind ribosome, proteins
Signals
start transfer peptide - binds wi SRP - starts translocation
o internal start transfer signal can serve as anchor
stop transfer peptide - anchors protein in membrane
o multipass transmem. proteins - internal start,
alternating stop & start seq.
ER resident proteins
o ER retention signal - K[)El (lys-Asp-Glu-leu)
o most complex - too large for most transport vesicle's
Some proteins imported post-translationally
post-translation translocation requires motor protein
o BiP - pulls in protein
Goigi (dictyosome)
cis - ER side, trans - plasma membrane side
Anterograde transport through the Golgi apparatus occurs
via cisternal progression
Peroxisomes
catalase & urate oxidase (crystallized core)
o catalase - hydrogen peroxide -> water + oxygen
o use O
2
to remove H from organic substrates (13
oxidation)
Import
o peroxins - import proteins into peroxisomes
o C-term peroxisome import seq. - Serine-lysine-leucine
o Unknown N-term sequence
Iysosomes - acid hydrolases
hydrolyzes cholesterylesters
o releases cholesterol & fatty acids into cytosol
mannose 6-phosphate (M6P) - targets proteins for
Iysosomes
endosomes - acidic (H+ ATPase)
early, late recycling - recycles & stores membrane proteins-
receptors, etc
Multivesicular bodies (MVBs) - endosome wi invaginations
sorting into internal vesicles regulated by ubiquitin
hyaloplasm - cytosol
tonoplast - single-membrane of vacuole
Prokaryotic pseudo-organelles
chlorosome - photosynthetic membrane invaginations
mesosome - invagination to which DNA is attached
GAMETES
primordial germ cells (PGCs)
cells singled out early on gamete progenitors
Sperm development
SRY (sex-determining region of Y) - directs testis development
Sertoli cells - main support cells in testis
sperm cells
greater quality control than egg development
contribute more mutations (larger number of mitotic divisions)
acrosomal vesicle - secretory vesicle at head of sperm
syncytium - mUltiple nuclei wi shared cytoplasm
spermatogonium -(rep.)-7 prim. spermatocyte -(mei-l)-7 sec.
spermatocyte -(mei-II)-7 spermatid - spermatozoa (mature sperm)
oogonium -(rep.)-7 prim. oocyte -(meiOSiS 1)-7 sec. oocyte -(mei-
11)-7 oovid - ovum (mature egg)
egg cells
main source of aneuploidy
parthenogenetic - eggs activated in absence of sperm
nurse cells - feed mRNA into oocyte
Fertilization - usually egg is a secondary oocyte
(1) capacitation - sperm modif. by female reproductive tract
o changes in glycoproteins, lipids, ion channels & RP
o unmasks cell-surface receptors that help sperm bind
o greatly increases flagellum motility
. (2) reach site of fertilization in oviduct
(3) migrate through granulosa ~ l l s surrounding egg
(4) bind to & cross zona pellucid
(5) bind to & fuse wi plasma membrane
o fusion activates egg by increasing Ca
2
+ in cytosol
o sperm donates centrioles (lacking in unfertilized egg)
polyspermy - more than one sperm fuses
polyspermy blocks
fusion of first sperm causes change in egg plasma membrane
cortical reaction - enzymes change structure of zona pellucid
o second polar body is extruded
aneuploid - cells wi abnormal chromosome #
euploid - cells wi normal chromosome #
in vitro fertilization (lVF) - aids infertile women
intracytoplasmic sperm injection (lC51) - aids infertile men
Inheritence
genomic imprinting - expression of maternal or paternal gene only
epigenetic inheritance - inherited protein structure superimposed
on genetic (DNA-based) inheritance
maternal effect - accumulated mRNA of mother's genes that
dictates early development
OTHER CELL TYPES
Stem Cells
Depend on contact signals from stromal cells
totipotent - any cell type, including extraembryonic
pluripotent - any cell type, but extraembryonic cells (placenta)
immortal strand hypothesis - stem cells maintain same DNA
transit amplifying cells - divide frequently, leave basal layer
terminal cell differentiation - final specialized characteristics
cellysually permanently stops dividing
apical meristems - undifferentiated pluripotent growing tip in plants
Epithelial cells
keratinocytes - epithelial cells wi excessive keratin intermediate
filament synthesis which give epidermis its toughness
{'J
Bone Cells
osteoblasts - construct bone
osteocyte imprisoned in hard matrix
osteoc/asts - remove bone (controlled by osteoblast signals)
Liver cells
hepatocytes
abundant smooth ER (principle site of lipoprotein production)
differentiated cells, no apparent stem cells .
interface between digestive tract and blood
major function - maintain blood glucose level (G6Pase) , ..
Lungs
alveoli - air-filled sacs of lungs
Type 1- most of wall, thin & flat to allow gas exchange
Type /I interspersed among Type I, plump, secrete surfactant
defensins
antimicrobial peptides that kill in mucus layer
most abundant proteins in neutrophils
short (12-50 AAs) & positively charged
Digestive tract
goblet cells, ciliated cel/s, & macrophages - keep airways clean
goblet cells - secrete mucus
absorptive cells (brush-border cells or enterocytes)
microvilli increase nutrient uptake & secrete digestion enzymes
enteroendocrine cells
secrete serotonin & peptide hormones that act on gut wall
paneth cells - innate immune cells in small intestine
similar to neutrophils, secrete cryptdins (defensin family)
stem cell niche crypt in gut lining that protects stem cells
Digestive acids & enzymes
gastrin - stims secretion of HCI into stomach, optimal for pepsin
HCI acid - low pH acts as a denaturing agent
pepsin - (chief cells) cleaves phe,leu(not next to proline}
chymotrypsin - cleaves tyr, phe, leu, lie, val, trp, & his at high pH
trypsin - (duodenum) cleaves lysine & arginine (not next to proline)
BLOOD
Blood buffering
Carbonic anhydrase CO
2
+ H
2
0 -> H+ + HC0
3
-
Blood-related cells
endothelial cells - form linings of blood vessels & lymphatics
platelets - cell fragments derived from megakaryocytes
pericyte (rouget cell, mural cell).- blood vessel support cells
differentiate into fibroblast, smooth muscle, or macrophage
Blood-related transport proteins
transferrin - carries iron in the blood
serum albumin carries fatty acids to heart & skeletal muscle
Red Blood cell promoting hormones
colony-stimulating factors (CSFs)
support hemopoiesis (formation of blood cellular components)
stims
o erythropoietin stims production of erythrocytes
o interleukin-3 (lL3) - promotes survival & proliferation of
earlier erythroid progenitor cells
Blood cell lineage
BFU-E
myeloid stem cell
pluripotent hematopoetic stem cell (hemocytoblasts)
Reticulocyte - immature erythrocyte
Erythrocyte - red blood cell
Hemoglobin
heme - porphyrin ring, synth'd from
R state - relaxed (higher O
2
affinity) / T state - tense (lower O
2
affinity)
O
2
binding
o moves heme iron closer to porphryn ring plane
o prevents binding of 2,3-bisphosphoglycerate (2,3-BPG)
lowers O
2
affinity
o converts T to R state
H+ & CO
2
binding
o binding inversely related to O
2
, stabilizes T form (Bohr
effect)
o H+ - binds to several places.
o CO
2
- binds a-amino at N-term of each subunit
o transported from cells to lungs & kidneys
fetal hemoglobin - higher O
2
affinity than mother's adult hemoglobin
(due to decreased binding of 2,3-BPG)
Hemoglobin S - abnormal form responsible for sickle cell anemia
Mutant - (negative AA -> hydrophobic AA)
forms fibrous aggregates when O
2
released
Oxygen binding models
MWC model/concerted model - all subunits undergo
transformation simultaneously
Sequential model binding of one ligand makes binding of adjacent
ligands more likely
Hill plot -log[6/1- 6] v 10g[LJ
Hill coefficient - slope of Hill plot - a measure of cooperative
interaction between protein subunits
HORMONES
peptides, amine, eicosanoid hormones act at cell surface
steroid, vitamin 0, retinoid, thyroid horomes act on nuclear
receptors
Peptide hormones
leptin
produced by adipose tissue
regulates energy uptake & expenditure
acts on arcuate nucleous of hypothalamus - inhibits eating
sympathetic nervous system - uncouples mit ox ph ->
thermogenesis
functions thru Jak/STAT
ghrelin - produced in stomach - producesfeeling of hunger
adiponectin stim fatty acid uptake / oxidation, inhibits fatty acid
synthesis
functions thru AMPK
atriopeptin - atrial natriu retic peptide/factor (AN P / AN F)
hormone secreted atria to lower blood pressure
o reduces water, Na+, & adipose loads in blood
vasopressin kidneys reabsorb water
15
Steroid hormones
aldosterone - kidneys retain Na+& water
Essential fatty acids
mammals cannot introduce more than 1 double bond into fatfy
acids
obtained from plants
linoleate (C-18 cis-t,9, t,12}
a-linolenate (C-18 cis-t,9, t,12,t,lS)
linoleate -7 arachidonate (20-C cis-t,s, t,8,t,11,t,14} -7 eicosanoids
(prostaglandins, thromboxanes, potent signal"
moelecules
Eicosanoids
Prostoglandins - regulate synthesis of cAMP (mediate pain,
fever, and inflammation}
Thromboxanes mediates blood Clotting (produced by
platelets)
Leukotrienes powerful signal in aJlergicreactions
Brassinosteroids - regulate growth/differentiation in plants
Plant hormones (growth regulators)
Ethylene - gas molecule
o Fruit ripening, leaf abscission, plant senescence
o Stress signal wounding, infection, flooding
o Stem thickening, shields tip of root, changes growth
direction /ltriple response"
auxin - helps plants grow toward light, roots down
MUSCLES
myoblasts - precursors of skeletal muscle fibers
satellite cells - fuse to repair damaged muscle or allowfor muscle
growth
muscle cells
generally survives for entire animal lifetime (individual
nuclei can be added or lost)
sarcoplasmic reticulum - abundant, modified smooth ER
found in muscle
fast-twitch muscle - fewer mitochondria & less blood supply
than slow-twitch muscle, quicker to fatigue
phosphocreatine / creatine phosphate / phosphagens -
reservoir of high-energy phosphates
myostatin - secreted to limit own growth
nitric oxide relaxes smooth muscle (thru guanylyl cyclase
-7 cGMP -7 protein kinase G}
myofibril- basic contractile element
sacromere contractile unit
A band- thick filament
I band - only thin filament
M-line - bisects A band (thick filament} where myosin heads
switch polarity (para myosin, C-protein, M-protein)
Z-disk - CapZ & a-actinin - crosslinks & caps (+} ends of actin,
bisects I band (desmin}
tropomodulin - stabilizes minus end of actin
"molecular rulers"
o nebulin - template protein along length of actin
o titin - links myosin to CapZ
Contractile cycle
(1) ATP binds myosin, actin unbinds
(2} ATP hydrolyzed, conformational change & weak binding to
F-actin subunit
(3) Pi released, myosin bond strengthened
(4} pi release triggers "power strokelJ w/ ADP release
Contraction Regulation
tropomyosin- binds actin, blocking myosinattachhlent
troponin(T,I,C}
o Toponin 1-T pulls tropomyosin out of normal
binding groove - preventing myosin binding
o Troponin C-binds 1-4 Ca2+causing I to release
actin exposing myosin-binding sites
Neuromuscular transmission
, 1. nerve impulse causes uptake of Ca
2
+ (v-gated Ca
2
+ chan}
which stim release of acetylcholine
2. acetylcholine binds to nicotinic acetylcholine receptor, influx
of Na+ depolarizes muscle
. 3. v-gated Na + channel lets more Na + in
4. depolarization activates v-gatedCa
2
+ channels in transverse
tubules
5. causes ci+ gated Ca
2
+ release channels (Ryanodine
receptors) in SR to open - myofibrils in muscle cell contract
Fermentation (regenerates NAD+)
pyruvate -(NADH-NAD+}-7 lactate
pyruvate -> C02 + acetaldehyde -(NADH-NAD+}-7 ethanol
pyruvate -(lactate dehydrogenase}-7 lactate + NAD+
pyruvate -(pyruvate decarboxylase}-7 acetaldehyde-
alcohol dehydrogenase-> ethanol + NAD+
o generates molecule of CO
2
from carboxyl
group of pyruvate (carbons 3 & 4 of glucose)
acetaldehyde - builds up in liver from ethanol
Cori cycle
when muscles lack sufficient oxygen, pyruvate reduced to
lactate
lactate exported to liver
liver converts to pyruvate, then glucose & exported
Glucose-Alanine Cycle
in muscles: pyruvate + ammonia -7 alanine
alaninine transported to liver & converted to glucose & urea
alanine + a-ketoglutarate -7 pyruvate + glutamate -7 glucose
NERVOUS CELLS
neuroblasts - nerve-cell precursors
neurons
- use only glucose & l3-hydroxybutyrate for energy (13- .
hydroxybutyrate converted to acetyl-CoA viaE1\in brain)
-most ATP used for transport of Na+ & K+, and maintenance of
potential
glial cells - specialized supporting cells that form myelin
Schwanncells peripheral nerves
Oligodendrites ..:..central nerves
Neuron Ion channels
voltage-gated Na+ channels responsible for AP (present at Nodes
of Ranvier along axons)
Ib
rapidly inactivating K+ channels - reduce AP firing at levels just
above that needed for stimulation
results in firing rate that is proportional to strength of
depolarizing stimulus over a broad range
Ca
2
+-activated !( channel - allows for adaptation to signal
Glutamate-gated ion channels
AMPA receptors - carries most of depolarizing current
responsible for excitatory PSPs (post synaptic potentials)
NMDA receptors - double-gated -glutamate must be bound
& membrane must be strongly depolarized
o releases Mg2+
o Ml+ blocks resting chanhel, Ca
2
+ in
Long-term potential (LTP) high activation & Ca
2
+ levels
activate protein kinases
Long-term depression (LTD) modest activation & Ca
2
+
levels activate protein phosphates
Resting ion concentrations
low inside/high outside
- K+ low outside/high inside
- Ca2+ low inside/high outside
- Cl- low inside/high outside
Axon Growth & Guidance
neurotrophic factors released by targets guide nerve growth
cone
Eph to Ephrin, Ephrin to Eph, Semaphorin to Plexin
MEMBRANES & TRANSPORT
Membrane Solubility
Highly Soluble
hydrophobic molecules (0
2
, CO
2
, N
2
, steroids, hormones)
uncharged polar molecules (H
2
0, urea, glycerol)
Low to no solubility
large uncharged polar molecules
ions
Membrane Composition
- phospholipids asymmetrically distributed between layers
o helps w/ signaling, binding, living/dead discernment
- ffippase (p-type pump) - flip-flop diffusion
o moves phosphatidylserine &
phosphatidylethanolanine to cytosolic leaflet
- lipid rafts - high conc. of sphingolipids, cholesterol & proteins
Endocytosis
Pinocytosis - endocytosis of small parts of membrane for
recycling
Caveolae - caveolins - also capable of endocytosis
Vesicular Transport
Coated vesicles
Contain
Cargo proteins
Cargo protein receptors
Coat-recruitment GTPases
o Arf proteins - clathrin & COPI assembly in Golgi
o SarI proteins COPli assembly in ER
Coat
COPI-coated - from Golgi
CGPII-coated from ER
clathrin-coated -Golgi <-> plasmal membrane <:->
endosomes
o coated pits - endocytosis
PIPs (phosphoinositrides) mark organelles & membrane domains
dynamin - forms spiral around neck of bud - helps break. off buds.
Rab proteins - guide vesicle targeting, on transport vesicles & target
membrane
Rab effectors
o membrane tethering - tether first attaches to Rab,
directing vesicle
o motor protein - propel vesicle along actin filament
SNAREs - recognizes proteins for targeting to membs/organelles
v-SNAREs - vesicle
t-SNAREs - target
NSF factor) separates SNAREs
for reuse
secretory vesicles - concentrate cargo, activate by proteolytic
cleavage
synaptic vesicles - partially fused and primed for excretion, locally
recycled and reused
synaptotagmin - Ca
2
+ sensor, promotes rapid fusion of
docked synaptic vesicles w/ presynaptic membrane
ATP driven pumps
Hype - turbine like, powered by electrochemical gradients
P-type phosphyrylate themselves during pumping
(Na+,K+,H+,Ca2+ gradients)
o Ca2+ pump (Ca2+ ATPase) - sarcoplasmic reticulum
pumps Ca2+ into SR
o Na+-K+ pump (Na+ pump/Na+-K+ ATPase) -ATP driven
antiport Na+ out, K+ in
V-type (vacuolar) - proton pumps that acidify membrane-
bounded organelles (vacuoles, Iysosomes, & endosomes)
ABC transporters-small molecules - ATP-binding cassette
o Multidrug resistance (MDR) protein - pumps
hydrophobic drugs out of cytosol
Overexpression gives cancer cells ability to
pump out multiple drugs
Ion channels (Na+,K+,Ca
2
+,Cr)
voltage-gated
o Na+ - skeletal muscle + nerve Na+ down its gradient,
create Action Potential
o K+ (delayed K+ channels) K+ out (down gradient)
returns nerve to negative potential
mechanically-gated
ligand-gated
o transmitter-gated - convert extracellular chemical
signals to electric
excitatory neurotransmitters - open cation
channels, influx of Na+
inhibitory neurotransrnitters - open CJ- or K+
neurotransmitters are not exclusively inhibitory or
excitatory
17
primarily excitatory -acetylcholine, glutamate,
serotonin
inhibitory GABA (y-aminobutyric acid), glycine
o ion-gated, nucleotide-gated
Pumps, Channels & Transporters
Lactose transporter / permease proton-driven co-transporter
(symport)
Na+-H+ exchanger (antiport) - H+ out, Na+ in
Na +-driven Ci"HC0
3
- exchanger - Na+ & HC0
3
- in, cr & H+ out
Na+independent CrHC0
3
- exchanger - HC0
3
- out facilitates ql!ick
discharge of CO
2
in red blood cells' ,
Na +-K- pump - Na + in, K+ out
K+ leak channels - open even when unstimulated/resting
MFS (major-facilitator superfamily) - transport small solutes in
response to ion gradients
ionophores bind ions for transport across membranes
aquaporins - H
2
0 pore, high cone. in epithelial cells of kidney
RECEPTORS
Ion channel-coupled transmitter-gated ion channels ionotropic
receptors
G-protein-coupled - GTP-binding protein modulates interaction bin
receptor & target
Most cell-surface receptors in animals are GPCRs
Enzyme-coupled - function as enzymes or associate w/ enzymes
Orphan nuclear receptors - receptors w/ unknown ligands
Second messengers
cyclic AMP, Ca
z
+, diacylglycerol
G-protein-coupled receptors (GPCRs) largest family of receptors
Stimulatory G-protein (G,l activates adenylyl cyclase
Inhibitory G-protein (G;) inhibits adenylyl cyclase +
regulates ion channels
ct subunit - binds GDP & GTP
GRKs (G-protein receptor kinases) - desensitize/inhibit
GPCR
Examples
rhodopsin receptor
6-adrenergic receptor
o - receptor kinase
pps
binds to pp, triggers receptor
sequestration
acetylcholine receptor
. 0 muscarinic acetylcholine receptor (in heart muscle)
opening of K+ channels
Pathways
G
s
adenylyl cyclase cAM PKA CREB + CREBB
DNA-binding
G
i
inhibits adenylyl cyclase & ion channels
G
q
--7 phospholipase IP
3
(inositoll;4,S-
trisphosphate) + diacylglycerol (DAG) fcleaved from PIPzl
o IP
3
--7 IPrgated Ca
2
+ release channels (ER) +
calmodulin CaM kinase CREB + CREBB DNA-
binding
o DAG arachidonic acid eicosanoids
prostaglandins (pain & inflammatory response)
Enzyme-coupled receptors
Most planet receptors are enzyme-coupled
receptor tyrosine kinases (RTKs) - growth factors
o most enzyme-coupled in animals are RTKs (rare in
plants)
o ephrins - cell surface bound extracellular signals
(bidirectional signaling)
o Eph receptors - cross-phosphorylation - creates docking
sites for signaling proteins (phospholipase C-y)
o Src & PI 3-kinase (phosphoinositide 3-kinase) - pps PH
(pleckstrin homology) where signaling complexes
assemble
o SH2 & SH3 - phosphotyrosine-binding (PTB) domains
tyrosine kinase-associated receptors depend on tyrosine
phosphorylation but lack kinase domain
o antigen & interleukins receptors in lymphocytes
receptor serine/threonine kinases
o most common receptor in plants - LRR (leUcine-rich
repeat receptor kinases)
o tetrameric receptor complex of dimer'd type I & type II
histidine kinase-associated receptors
o two-component signaling pathway
o chemotaxis receptors - methylation responsible for
adaptation
receptor guanylyl cyclases
receptorlike tyrosine phosphatases
Desensitization
receptor sequestration - receptors removed from PM to
endosome(s)
receptor down-regulation - receptor destruction in
Iysosome(s)
receptor inactivation cytosolic domain is blocked
signaling protein inactivation intracellular signaling protein
is inactivated
production of inhibitory protein - pathway produces protein
that inhibits future signal
TRANDUCTION PATHWAYS
SMAD pathway
inhibits excess cell proliferation
(transforming growth factor
receptor II + receptor I Smad3 + Smad4
DNA-binding
signal receptor II to pp receptor f
receptor I pp's Smad3
pp'd Smad3 binds Smad4 & is imported into nucleus
Inhibitors I-Smad, Ski, SnoN
Wnt signaling pathway
Wnt secreted signal proteins ( local mediator/morphogen)
Frizzled cell-surface receptors
Disheveled scaffold protein
multifunctional protein
Wnt LRP + Frizzled Disheveled -I destruction complex
catenin -I Groucho DNA-binding
lRP & Frizzled binding of Wnt activates Disheveled
Disheveled inhibits destruction complex (GSK-3)
inhibits Groucho, allowing LEF binding of DNA
JAK-STAT signaling pathway
cytokine receptors - dimers or trimers
JAKs - Janus kinases
STATs - gene regulatory proteins
cytokine -7 cytokine receptor -7 JAK -7 STAT-binding -7 pSTAT-7
2xSTAT -7 DNA-binding
cytokine brings JAKs together - pps each other, pp cytokine
receptor
STAT docks to pp'd cytokine receptor & pp'd by JAKs
pp'd STATs dimerize & enter nucleus
MAPK/ERK pathway
- RTK (receptor tyrosine kinase)
o Insulin receptor
o EGF receptor (epidermal growth factor)
- Ras - GTPase, acts as signaling hub
MAPK - mitogen-activated protein kinase
RTK -7 Grb2 -7 RasGEF (SOS) -7 MAPKKK (Raf) -7 MAPKK {Mek)-7
(MAPK (Erk) -7 gene regulatory proteins or protein kinases
Notch signaling pathway
conserved developmental pathway
Delta signal- tells adjacent cells not to be neural
lateral inhibition - neurons inhibit growth of
additional/redundant neurons
Delta -7 Notch receptor -7 TACE & Presenilin 1 -7 Notch segment
-7
SENSORY SIGNALING
Sight
light activation of rhodopsin receptor
rhodopsin
o chromophore ii-cis retinal-light-absorbing
pigment
o opsin - protein portion
ll-cis-retinal is converted to all-trans-retinal
Ught -7 rhodopsin -7 Gt (transducin) -7 cGMP
-7cGMP-gate ion channels close
-7AP
guanylate cyclase converts GTP to cGMP
arrestin - binds phosphorylated rhodopsin, inhibiting it
retina contains - 10
9
rod cells & 3 x 10
6
cone cells
blue, green, & red cones distinguished environment of not
difference in absorbing molecule
Smell
olfactory receptors (GPCR) - Golf - cAMP - cyclic-AMP-gated cation
channels - Na+ influx
Pain
Nocireceptors pain receptors that respond to heat, acidity,
mechanical change
Taste
passage of ions thought responsible for taste sensations
gustducin - G protein associated w/ basic taste
Sound
ability to detect range of frequencies due to distinctions in basilar
membrane
PLASTIDS, PHOTOSYNTHESIS
Proplastids - no internal membranes or chlorophyll
chloroplast - photosynthetic organelle
chromoplast - pigment organelle
leucoplast - storage plastid
o amyloplast - stores starch (amylopectin) granules
Chloroplast
thought to have originated from cyanobacterium
thylakoid membrane -light absorption & electron transport
stroma chloroplast matrix
granum(a)- stack(s) of thylakoids
make most of own lipids & some amino acids
mitochondria & chloroplasts are maternally inherited in
2/3rds of higher plants
o in some plants, inheritance is biparental
Neither ATP nor NADPH gen'd in chloroplast cancross
chloroplast membrane
Photosystems
LHCs -light harvesting complexes
exciton - quantum of energy passed from excited molecule
to another
spatially distinct, located in thylakoid membrane (lamellae)
o PSlllocated on appressedside
o PSI located on nonappressed side
chlorophyll- Mg2+ held in porphyrin ring
o P
680
and P
700
- specialized chlorophyll molecules located in
"reaction centers"
2 photosystems required to evolve O
2
(oxygenic
photosynthesis)
o plants, algae, & cyanobacteria - contain both PSI and PSII
o purple bacteria and green sulfur bacteria w/ only 1
photosystem, cannot evolve O
2
Photosystem 11- Type II (Pheophytin-QuinoneJ Reaction Center
most similar to the bacterial reaction center
higher energy to excite (shorter wavelength)
Structure
OEC (Oxygen-evolving/water splitting complex)
o 2 H
2
0 -> 4H+ + 4e- + O
2
o passes electrons to P680
o Manganese center/complex - binds water
cytochrome b6-f complex - pumps H+ into thylakoid
space
pheophytin - chlorophyll lacking central Mg2+ ion
P680 -> pheophytin -> Po,. ->PQB (plastoquinones) -> cytochrome
b
6
f complex ->PQ
s
H
2
-> cytochrome f -> -> PSI

Photosystem 1- Type I (Fe-S) Reaction Center
does not contribute to proton gradient
generates NADPH (NADP+ from stroma)
P700 -> A0 (Ao-) -> A1- (phyllquinone) -> 3x -> Fd (ferrodixin)+
NADPH + H+
Noncyclic photophosphorylation
- 2 photosystems working in serial
PSII -> plastoquinone -> cytochrome b6-f complex -> plastocyanin ->
PSI -> ferredoxin -> ferredoxin NADP reductase
Cyclic photophosphorylation
- electrons cycle thru PSI reaction center
- ATP formed w/ no net NADPH or O
2
'
- PSI -> cytochrome b
6
f complex '
Chromatophores
Carotenoids
- accessory pigments that extend light absorption range
- protect against damaging photochemical reactions & reactive
oxygen species
- 8-carotene red-orange isoprenoid
- lutein yellow blue light absorption
ph ycobilisome
-light-harvesting antennae of PSI! of cyanobacteria & red algae
Bacteriorhodopsin
-light-driven proton pump only 247 amino acids long
Phototrophic sensory system
- allow plants to respond to light
- phytochromes - cytoplasmic ser/thr kinases red light
- phototropin - blue light
- cryptochromes - flavorproteins sensitive to blue light
CALVIN CYCLE & PHOTORESPIRATION
Calvin cyCle (carbon-fixation/assimulation)
- occursTn chforoplaststroma
3 C02 + 6 NADPH + 9 ATP + water -> glyceraldehyde 3-phosphate
Photosynthetic carbon reduction
(C0
2
-7-7-7 glyceraldehyde 3-phosphate)
Reactants Products
Step (3) CO
2
+ (3) ribulose 1,5 (6)
1 bisphosphate
Step (6) 3-phosphoglycerate + (6) 1,3-bisphosphoglycerate +
2 (6)ATP (6lATP
Step (6) 1,3- Glyceraldehyde 3-phosphate +
3 bisphosphoglycerate (6) NADP+ + (6) Pi
f>Jt\/)f .
Step (5) glyceraldehyde 3- (3) ribulose 5-phosphate + (2)
4 phosphate Pi
Step (3) ribulose 5-phosphate + (3) ribulose 1,5-bisphosphate
5 (3)ATP (first stable product)
Ribulose Regeneration (8 enzymes)
triose phosphate isomerase
phosphopentose isomerase
phosphopentose epimerase
fructose-1,6-bisphosphatase
sedoheptufose-1,7-bisphosphatase (unique to plants)
phosphoribulokinase (unique to plants)
aldolase (used twice)
transketolase (used twice)
pentose phosphate pathway & calvin cycle
Connects pentose phosphate pathway to glycolysis in animals
necessary for production of NADPH
fructose 6-phosphate + glyceraldehyde 3-phosphate
erythrose 4-phosphate + xylulose 5-phosphate
sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate
ribose 5-phosphate + xylulose 5-phosphate
Light Regulation of Calvin cycle
disulfide bondsformthat immobilize catalytic activity
Enzymes
Reduction
o ribulose 5-phosphate kinase
o glyceraldehyde 3-phosphate dehydrogenase - prevents
hexose synth competing w/ glycolysis in dark
Regenetation
o fructose 1,6 bisphosphatase
o sedoheptulose 1,7 bisphosphatase
RuBisCO (ribulose 1,S-bisphosphate carboxylase/oxygenase)
substrates - 0
21
C0
21
ribulose l,S-bisphosphate
requires 2 CO
2
for normal function (1
st
as substrate, 2
nd
for
binding site of metal ion cofactor)
Photorespiration
use of O
2
instead of CO
2
in Calvin cycle
caused when CO
2
is in low concentrations
wasteful use of energy w/o useful products
involves chloroplasts, peroxisomes, and mitochondria
rubisco's oxygenase activity
o ribulose l,5-bisphosphate -7 3-phosphoglycerate +
phosphoglycolate
carbons salvaged in glycolate pathway releasing
previously fixed CO
2
C4 plants (spatial separation)
isolates Calvin cycle to maximize CO
2
concentration
(minimizing energy & water loss of photorespiration)
mesophyll cells
o CO
2
+ phosphoenolpyruvate (PEP) -> oxaloacetate
-7 malate -(to bundle-sheath cells)
pyruvate phosphate dikinase - pyruvate -7 PEP
phosphoerolpyruvate carboxylase CO
2
+
phosphoenolpyruvate (PEP) -7
oxaloacetate
bundle-sheath cells
o malate -7 CO
2
+ pyruvate
CO2 -7 Calvin cycle
pyruvate + Pi + ATP -7 AMP + PP
i
+ PEP
(to mesophyll cells)
CAM (Crassulacean acid metabolism) plants (temporal separation)
-night - CO
2
fixed into malate & stored in vacuoles
-day stomata closed, malate source of CO
2
dihydroxyacetonephosphate
formed in stroma transports ATP out of chlo-roplasts (ATP
cannot be exported on its own)
transported by the Pi-triose phosphate anti porter
converted by glycolytic enzymes to 3-phosphoglycerate,
generating ATP in cytosol
3-phosphoglycerate reenters the chloroplast, completing the
cycle
glyceraldehyde 3-phosphate
turned into starch in stroma
turned into sucrose production in cytosol
Starch
amylose al-4linked planar polymer of glucose
amylopectin - alA link highly branched polymer of glucose,
al-6 branch every 24-30 residues
synth'd in chloroplasts & amyloplasts
o temporary storage chloroplasts of leaf cells
o long-term storage amyloplasts of colorless parts (seeds,
roots, & tubers)
starch synthase - adds glucose to reducing end (donated by
ADP-glucose)
o Glucose I-phosphate + ATP -j ADP-glucose
o ADP-glucose -(starch synthasej-j starch
ADP-glucose pyrophosphorylase
o regulates starch synthesis
o activated by 3-phospholycerate / inhibited by Pi
Sucrose
synth'd in cytosol of leaf cells
2 steps from + fructose I-phosphate
Cellulose
UDP-glucose -(cellulose synthase)-> cellulose
synthesis takes place in terminal complexes (rosettes)
rosettes simultaneously produce 36paraileiceHulosechains
IVIITOCHON DRIA
Most catabolic processes (fatty acid oxid,) occur in the mito. matrix
Cristae inner membrane invaginations
Protein translocators
TOM complex - transfer across outer mem
TIM complex - transfer acrOss inner mem - aided by memb potential
SAM complex - helps jl-barrels fold in outer membrane
OXA complex - moderates insertion of inner mem proteins that are
synthesized inside mitochondria
Mitochondrial Hsp70 - acts as motor to pull precursor protein into
matrix f?,;F I,,,
Stop-transfer sequence prevents further translocation across inner
membrane
Mitochondrial genome
Human mitochondria encode own ribosomal RNAs
-Plant mitochondria encode 5S mitochondrial rRNA, uses
standard genetic code
Mito. membrane
most lipids imported & modified rather than synthesized
high percent of lipids converted into cardolipin (4 fatty acid
tails)
impermeable to NADH (shuttle systems carry in reducing
equivalents)
proton gradient active transports:
o in pyruvate, Pi, ADP
o out ATP out
3 protein types
o electron transport chain
o ATP synthase
o metabolite transport proteins
Electron Transport (Respiratory) Chain
# Name Reaction e' carriers Notes
+
NADH dehydrogenase
NADH -7
FMN, FeS
ubiquinone
succinate-Q reductase / succinate -7
FAD, FeS
not involved in
dehydrogenase ubiquinone proton gradient
ubiquinone - mobile carrier- (synth'd from benzoquinone [tyrosine] &
isoprene {acetyl-CoAl
Cytochrome bCl
hemes,
ill',
complex ubiquinone -7 Rieske - 2 His
CoQ cytochrome c cytochrome c
FeS, Rieske
instead of Cys
oxi do red ucta se
ISP
cytochrome c - mobile carrier - heme group, role in apoptosis
IV cytochrome c oxidase
cytochrome c -7 hemes;
O
2 CUA' CUB
ATP synthesis
Mitchell chemiosmotic hypothesis - e' transport generates
proton-motive force that provides energy for ATP synthesis
Location - mito. Matrix, chloroplast stroma, cytosol in proks
A TP synthasome
o ATP synthase
Fl - ATPase head + rotor stalk
Fo - transmembrane H+ carrier / proton pore
o adenine nucleotide trans/ocase (A TP/ADP antiporter) -
exchanges ATP/ADP (ADP availability limits synthesis)
o phosphate translocase symports H
2
P0
4
- & H+ into
matrix
o therrnogen (uncoupling protein) - energy of oxidation
not conserved as ATP, but dissipated as heat
Shuttles
malate-aspartate shuttle (liver, kidney, heart) 2.5 ATP
NADH + oxaloacetate -j NAD+ + malate -j oxaloacetate +
NADH
reducing equivalents from cytosolic NADH transferred to
malate (malate transported & replenishes NADH)
ions from malate, not malate transported across inner mito.
memb:-r
shuttle (skeletal muscle + brain) 1,5 ATP
NADH to Complex III
converts reducing equivalents directly to ubiquinone in inner
mito. memb.
Oxygen Radicals
superoxide dismutase - antioxidant in cytosol, mitochondria &
extracellular (2 '0
2
" + 2H+ -7 H
2
0
2
+ O
2
)
glutathione peroxidase reduce lipid hydroperoxides & hydrogen
peroxide (H
2
0
2
-j H
2
0)
CYTOSKELETON
Actin
Form
21
examples: contractile ring (myosin II), muscle (myosin II),
stereocilia, microvilli
bindsATP/ADP
o Tform -grow (ATP)
o D form - shrink (ADP) [cofilin - causes D-form actin
disassembly]
plus end - faster polymerization & depolymerization
actin capping - dramatically lowers critical concentration &
increases growth rate
Actin nucleation
Nucleated at cell cortext (just beneath PM)
ARPcomplex
nucleates most effectively when bound to existing actin
filament
branch from preexisting filament by
formin - nucleate actin growth by capturing two actin monomers,
follows along plus end
Rho family - subfamily of Ras, regulate many aspects of actin & MT
dynamics (thru ARP & formin)
bound to bound to GDls (guanine nucleotide dissociation
factors) when inactive
play part in cell proliferation, division, gene expression, &
apoptosis
Cdc42 - affects filopodia
Rac1- affects lamellipodia
RhoA - affects stress fibers
Cross-linking proteins
ERM family - mediates interactions between actin & PM
dystrophin - connects muscle fiber to EM thru PM
spectrin dimers
Actin arrays
contractile bundle - t1-actinincrosslinks
gel-like network - filamin crosslinks - important for cell
motility
tight parallel bundle - fimbrin crosslinks
Motor proteins
myosin superfamily
o Structure:
2 heavy chains a-helixes coiled-coil + 4 light chains
(51 fragment - myosin head)
N-term - head/motor domain
C-term tail
motor activity Regulated thru pp'tion
Plasma Membrane protrusions
Activity
Types
actin filaments bend
arp2/3 nucleation of new filaments
actin depolymerization & polymerization
Filopedia - 1D projections
Lamellipodia - 2D - sheet-like extensions
Pseudopodia - 3D
Regulation
Rac superfamily (related to Ras) stims formation of
lamellipodia & stress fibers
o Activated by growth factors
Microtubules
Form
Consist of protofilaments made up of tubulin
a-tubulin & - dimerize
o a oriented toward oriented toward (+)
o u-boundGTP intra-linksdimer (cannot be lost)
o !,-bound GTP inter-linksdimers (can be lost)
Microtubule Nucleation
critical concentration of tubulin, energy (37"C), & GTP
MTOC (microtubule organizing center)
y-TuRC ring complex) nucleates microtubule
centrosome (single well defined MTOC in animals)
centrioles - cylinders arranged in L shape at centrosome
center
centrosome matrix/pericentriolar material
spindle pole body (MTOC in fungi & diatoms)
imbedded in nuclear membrane
microtubules nucleate all aroundnuc/eus in plants
Microtubule motor proteins
Kinesin mostly toward (+) - 2 heavy chains, 2 light chains
one family C-term head, walks toward (-)
o Hand over hand motion - never completely dissociates
o Nonclaret disjunctional(NCD) -toward (-) kinesin
Dyneins - toward (-) - 2-3 heavy chains + variable
intermediate & light chains
o Largest & fastest molecular motors
dynactin protein complex
links cargo (organelles, chromosomes) to dynein & kinesin
Associated proteins
MAPs (microtubule-associated proteins) bind alongside MTs
mediate interactions b/w MTs & other components
Tau & MAP2
plectin - crosslinks actin, MTs & intermediate filaments
catastrophe factor- increases chance of microtubule catastrophe
Mitotic Spindle
Centrosome - spindle pole
MTs
o astral- interact w/ cell cortex - dynamic instability
o kinetochore - attach each chrom to each spindle pole
o interpolar - hold halves of spindle together by overlapping
Kinetochores attach MTs to chromosomes
in animals, position of contractile ring determined by signals from
anaphase spindle
poleward flux - net addition of tublin to (+), loss at H
average lifetime of microtubules during mitosis is 60-90 seconds
Ran-GTP & GEF bound to chromatin, stabilize MT spindles
Cilia & flagella
Axoneme 9 doublet MTs (A-full, B-partial) arranged around
2 MTs
Dynein responsible for MT sliding during flagellar bending
Flagella != bacterial flagella (made up of flagellin, not MTs)
Basal bodies (kinetosome) firm root of most cilia & flagella
Neurons - single MTs do no extend over the length of
axons/dendeites overlap
Dendrites - MTs of mixed polarity
Intermediate filaments
Form
- _ tetrameric,disassembly triggered by subunit phosphorylation
Types
keratins - epithelial cells (hair, horns, etc)
o high % of glycine, alanine, & cysteine (14%)
o heterodimers of type I (acidic) & type II (neutral/basic)
o important in cell adhesion (in desmosomes)
desmin - heart muscle
neurofilaments - highly concentrated in axons
CELL CYCLE
Karyokinesis - nuclear div w/o cytoplasmic
"
dephosphorylation of H1 & Rb limit replication to once per cell cycle
Checkpoints
Start/restriction point - replication commitment (during G
I
)
G2/M checkpoint - triggers events leading to chromosome
alignment
spindle assembly checkpoint (metaphase-to-anaphase transition)
sister-chromatid separation (unattached kinetochores inhibit
separation)
Go - specialized nondividing state
terminally differentiated Go - cell-cycle control system
completely dismantled
Early in G1
assembly of pre-replication complexes at origins
degradation of S-phase cyclin-Cdk inhibitor
activation of pre-replication complexes
APC/C activity
o degradation of securin
o degradation of mitotic cyclins
Late G1
Cdc1 binds to APC/C - prevo inhibited by pp by M-cyclin-Cdk
Cdc1-APC/C activator
CKI accumulation + decr. cyclin expression suspend Cdk activity
G1/S-cyclins - trigger progression through start/restriction point
S phase - ~ hours in mammals
M-cyclin
synth increases during G2 & M due to increased transcription
Activated by Cdc25 removing inhibitory pps (by Wee1)
M-Cdk - activated by dephosphorylation at onset of mitosis
activates Cdc25 & inhibits Wee1
Induces assembly of mitotic spindle
Insures attachment of sister chromatids to both poles of
spindle
Triggers chromosome condensation
Promotes breakdown of nuclear envelope (in animals)
Aided by pole-like kinases & aurora kinases
Anaphase-promoting complexjcyclosome (APC/C)
ubiquitin ligase
Activating subunits
Cdcl ~ activation during late mitosis & early G1
o
o
inactivates Cdks & destroys geminin (allowing for
pre-RC assembly)
pp'd by M-Cdk, allowing Cdc20 to bind
Cdc20 - activating subunit of cyclosome during anaphase
(increased transcription as cell a'pproaches mitosis)
o Targets securin, S & M-cyclins for destruction (loss
of most Cdk activity)
Cdk Regulation
phosphyrlation of Cdk-subunit
o Cdk-activating kinases (CAK) - fully activates cyclin-
Cdk complex
o Cdc25 - removes inhibitory pp'tion, increaSing Cdk
activity
Binding of CKls (Cdk-inhibitors)
o Wee1- inhibits cyclin-Cdk complex
Proteolysis of cyclins
Changes in gene expression of Cdk regulators
Securin - binds to and inhibits separase - protease which cleaves
cohesion
Control of Cell division & Growth
Mitogens - stim Cl/S-Cdk activity & cell division
o Ras (GTPase) - activates MAP kinase cascade - Myc &
other gene reg. proteins
o G1-> G1-Cdk -> E2F proteins - G1/S-cyclins / S-cyclins /
DNA synthesis
Growth factors - stim cell growth (increase in mass) -
promotion of synthesis & inhibition of degradation
o TOR - stims S6 kinase, elF4E (initiator factor)
o Promotes gene expression of ribosomal subunits
Survival factors - suppress apoptosis
DNA damage-7 ATM/ATR kinase -7 Chk1/Chk2 kinase -7 p53 -7 p21
binds G1/S-Cdk & S-Cdk
Mdm2 - degrades unphosphorylated p53
Stages of M Phase
(1) prophase - chroms condense, spindle assembles bin
centrosomes
(2) prometaphase - breakdown of nuclear envelope, chroms attach
to spindle
(3) metaphase - chromosomes align at equator, sister chromatids
attached to opposite poles
(4) anaphase - chromosome segregation - synchronized separation
of sister chromatids
anaphase A - shortening of kinetochore MTs & movement of
chromosomes along MTs towards poles
anaphase B - sliding & elongation between interpolar MTs,
pulling force of astral MTs pull centrosomes apart
(5) telophase - chroms arrive at poles & decondense, nuclear
envelopes develop
o depp of proteins responsible for reassembly of nucleus
(6) cytokinesis - contractile ring (actin + myosin) divides cytoplasm
Prophase I
leptotene - homologs condense & pair, recombination begins
zygotene - synaptonemal complex develops at recombination sites
pachytene - assembly complete, homologs synapsed along entire
length (can persist for days or longer)
diplotene disassembly of synaptonemal complexes & chromosome
condensation (visible chiasmata, lambrush most active)
diakinesis - transition to metaphase I, thromsdetach from nucleus
interkinesis - period blw meiotic divisions
Preprophase band - marks site of cell wall assembly in plants
Phragmoplast - precursor to cell wall formed on interpolar MTs in
plants
APOPTOSIS
Procaspases - inactive precursors proteolytically cleaved by other
caspases
cleavesd in two spots large & small subunit - dimerize & form
tetramers
Prodomains I caspase recruitment domain - discarded
Caspases active site & cleave at aspartate (c-asp-ases)
Initiator procaspases - initiation executioner procaspases
Executioner procaspases cleave other EPs & target proteins,
cleaves protein that inhibits endonuclease
lAPs (inhibitors of apoptosis) - inhibit caspases
Anti-lAP - bind to lAP preventing their interference wi
caspases
Extrinsic pathway
extracellular signaling proteins bind to death receptors
Fas receptor binds Fas ligand
procaspases bind to "death domains" of Fas forming DISC
(death inducing signal complex)
FLIP - competes w/procaspase for binding on death domains,
lacks proteolytic activity
Intrinsic pathway
release of mitochondrial proteins into cytosol some activate
proto lytic caspase cascade
Cytochrome c - binds to adaptor protein Apafl (apoptotic
protease activating factor-i)
Apoptosome heptamer of cytochrome c + Apafl
Bel2 proteins regulate release of cytochrome c
BHl-4 anti-apoptotic, binds 1-4 (Bcl2 homology 1-4)
Bcl2 & Bcl-XL on cuytosolic surface of mit.mem.
BH123 pro-apoptotic, binds 1-3 - aggregate to pump out
cytochrome c
Bak - bound to cytosolic surface of mito.mem.
Bax - cytosol, transferred to Mito.mem. when active
BH3-only pro-apoptotic, inhibit anti-apoptotic
proteins
P53 - stims expression of BH3-only genes
Survival factors
stim expression of Bcl2
Uninhibit Bcl2 from BH3-only (Bad, etc)
Uninhibit lAPs (pp'ing anti-lAP (Hid, etc)
Sphingomyelinase (SMase) - cleaves sphingomyelin into ceramide,
thought to be part of apoptosis
pyknosis - condensed appearance of DNA indicating apoptosis
Necrosis
Cell swells wi water & ruptures
turmor necrosis factor -7 diacylglycerol -7 sphingomyelin
breakdown
CELL ADHESION
Cell-Cell Anchoring Junctions
Junction name Internal External
adherens cadherin actin
desmosomes cadherin intermediate filament
Cadherins - in animals, not plants or bacteria
- Depender:lt on ci+
- Generally like-like binding
adhesion belt - cad herins anchor proteins + actin
Desmosomes contain the transmembrane cadherin proteins
desmoglein and desmocollin
Occluding Junctions
I Junction name Organisms Proteins
I Tight junctiorl vertebrates claud in & occludin
I Septate junction invertebrates
fectins proteins that can bind carbohydrates, read sugar code
- sefectins - mediate cell-cell recognition
Immunoglobulin superfamily proteins mediate ci+ independent
cell-cell adhesion
- ICAMs intracellular cell-adhesion molecules
VCAMs - vascular
NCAMs neural
Junctiional complex - series of junctions between epithelial cells
tight, adherens, desmosomal
Signal-relaying junctions
Gap junctions - connect cells electrically & metabolically
o Connexins
o Coordinate activities of adjacent cells through sharing of
metabolites & ions
Synchronize muscle contractions
Important part of embryogenesis
o Permeability regulated - dopamine & Ca2+ (reduce or close)
Plasmodesmata - desmotubule - continuous wi ER
Chemical & Immunological synapses
o Scaffold proteins - important for cell-cell adhesion at
synapses
Cell polarity - dependent on membrane-associated proteins: Par3 +
Par 6 (scaffold proteins) & atypical protein kinase C (aPKC)
Cell-Matrix Anchoring Junctions
Junction name Internal External
actin-linked matrix integrin actin
adhesions
Hemidesmosomes integrin intermediate fila ment
Integrins
- mUltiple forms from multiple genes
- a + 13 subunit clamps onto matrix
talin - binds integrin to actin
Fibronectin - glycoprotein that aids in matrix.-cell interactions
Signaling through Integrins
Anchorage dependence - cell proliferation & survival tied to binding
to matrix
Focal adhesion kinase (FAK) - cross-pp - docking site for
Serine/tyrosine kinases
Number of focal adhesions regulate level of adhesion/motility
Extracellular Matrix
Fibroblast
Synth & maintains EM
can differentiate into
o bone cell (osteoblast/osteocyte)
o fat cell (adipocyte)
o cartilage cell (chondrocyte)
o smooth muscle cell
Animal EM
rich in nitrogen-containing polymers
excreted largely by fibroblasts
Basal lamina
functions
o molecular filter - proteoglycan key
o cell segregation
o scaffold
"
o regeneration - tough, often remains inact, guides repair &
regeneration
components
o fibrous proteins (usually glycoproteins)
o laminin - 3 chains, shaped like cross
o Type IV collagen - ropelikesuperhelix
o nidogen
proteoglycans - glycosaminoglycans (GAGs) + core proteins
perlecan crosslinks EM
GAGs (Glycosaminoglycan) - unbranched polysaccharides composed
of repeating disaccharide units (1 an amino sugar)
o Hyaluronan - simplest GAG, spun out directly from cell
surface by embedded enzyme complex
Collagen
major proteins of EM
exported from ER as folded triple helix
repeating tripeptides (glycine-x-y) rich in glycine, proline &
hydroxyproline
mature collagen does not contain cysteine or disulfide bonds
Types
o fibrillar collagens - Type I principle coHagen of skin & bone
o Fibril-associated collagens -IX & XII- retain propeptides-
organize fibrils
o Network-forming collagen -IV
o Anchoring fibrils - VII
Elastic fibers elastin - hydrophobic
Covered by sheath of microfibrils that act as scaffolding-
fibrillin
Proteoglycans
heparansulfate - present in all animal cells
common sugars:
o N-acetyl-O-glucosamine
o O-glucuronic acids (O-xylulose 5-phosphate -7 pentose
phosphate pathway)
o L-iduronic acid
Matrix degradation
Matrix metalloproteases - depend on bound Ca
2
+ or Zn
2
+
Serine proteases - highly reactive serine active site
Plant Cell walls
almost no nitrogen
microtubular - "cortical array" determines orientation of
cellulose synthase
Primary cell walls
b thin, extensible walls of newborn plant cells
o cellulose microfibrils interwoven w/ network of pectic
polysaccharides
o Pectins - negatively charged branched polysaccharide
Secondary cell walls
o rigid wall formed by depositing new layers of matrix inside
old ones
o Lignin network of phenolic compounds
Calmodulin (CaM) - calcium-binding protein - binds 4 Ca
2
+
responsible for regulatory ability of ci+
EF hand - helix-turn-helix found in Ca-binding proteins
major classes
fibronectin, Ig-superfamily CAMs, P-selectins
CaM kinases I-IV (Ca
2
+/calmodulin dependent protein kinases)
serine/threonine kinase
phosphorylates self (memory)
DEVELOPMENT & DIFFERENTIATION
Hoxcomplex
important switches that control cell identities by controlling
transcription target genes
carries permanent record of positional information
controls anteroposterior axis
Mammals have 4 Hox complexes
Morphogens
long-range inducers that exert graded effects
Hormonal signals that shape development, imposing a
pattern a whole field of cells
Induces graded responses, new gene expression, differentiation
events, & change in cell fate
Embryological Development
Concepts
Synctium - cell w/ multiple nuclei
Cel/u/arization subdivision of synctium so that each cell has only
one nucleus
Structures
embryonic disk ectoderm + endoderm
Blastula - hollow ball of cells, with an internal fluid-filled cavity
inner cell mass gives rise to extra-embryonic structures-
hypoblast & trophoblast
Blastopore - invagination that circle vegetal pole
Organizer (Spemann'sOrganizer) dorsal lip of blastopore
which organizes formation of main body axis
o of organizer-that defines central
body axis
gastrulation - transformation of simple ball or hollow sphere of cells
into structure with a gut
3 germ layers formed
ectoderm - exterior, precursor of epidermis and nervous
system
o neural tube
endoderm interior, precursor of gut and its appendages' (lung
& liver)
mesoderm cells blw ectoderm & endoderm; precursor of
muscles, connective tissues, etc.
o notochord defines primitive axis of embryo
o somites -segments (cohesive groups of celis, separated
by clefts)
form along neural tube dermis, skeleton, muscle
(heart)
Segmentation
metamerism - serially repeating segments
imaginal discs - groups of cells that are set aside, apparently
undifferentiated, in each segment of the larva
segmentation clock - repetitive alternating pattern of gene
expression
sequential induction - strategy for generating a progressively more
complicated pattern by repeating variations of few basic themes
Segmentation genes
gap - divides embryo into broad regions
pair-rule & segment polarity -defines segment stripes
homeotic - specifies which organs & appendages develop
Polarity
egg polarity - established as early as oogenesis in some species
Left-right polarity
o controlled by Nodal in embryo
Anterior-posterior patterning
o in limb bud Shh gradient
o in developing spinal chord - BMP, netrin, Shh
bicoid - anterior
nanos - posterior
CANCER
Types
Carcinomas - epithelial cells
Sarcomas - connective tissue
Leukemias - blood or bone marrow
Lymphomas -lymphocytes
Properties of cancer growth
more self-sufficient
o high glucose uptake & metabolism
o insensitive to anti-proliferative signals
o less prone to apoptosis
o induce help from stromal cells
o induce angiogenesis - formation of new blood vessels
o produce telomerase or other method to stabilize
chromosome length
o reduced growth factor requirement
altered morphology
o metastasize
o genetically unstable
o loss of actin microfilaments
o defective differentiation
o defective cell cycle braking
protein kinases are found to be overactive in cancer cells
Types of carcinogens
tumor initiators - damage DNA
tumor promoters - do not damage DNA
Cancer-critcal genes
proto-oncogenes - cancer risk from over activity - fos, myc,&
ras often growth factors and growth factor receptors
tumor suppressor genes - cancer risk from too little activity
DNA maintenance genes - mutation results in genetic
instability
Src - proto-oncogenic tyrosine kinases
Ras - g protein; mutated in 1 in 5 cancers
Rb - tumor suppressor
Myc - transcription factor I activator
Cancer Diagnosis
Intermediate filaments - may be used to diagnose tissue of origin
Ames test - assay to assess mutagenic potential
IMMUNOLOGY
pathogens
obligate pathogens - can only replicate inside the body of their host
and are called
facultative pathogens - replicate in water or soil, cause disease if
they encounter susceptible host
opportunistic pathogens - normally harmless, cause disease in
injured or immunocompromised host
vaccination - enhanced function of B cells & cytotoxic T cells
autoimmune disease - subverts immune system by destroying
helper T cells
transformation - uptake of naked DNA
transfection - introduction of foreign DNA by non-viral method
transduction - foreign DNA introduced via phage vector
conjugation genetic material transferred unidirectionally from
bacterial donor to recipient
recipient F
donors
o F+ piliated wi conjugative plasmid
o F' - conjugative plasmid contains chromosomal genes
o Hfr - conjugative plasmid incorporated into chromosome
rolling circle (sigma) replication
Bacteria
classified by shape (rods, spheres, spirals) & Gram-staining
properties
o Gramcpositive - single membrane & thick cross-linked
peptidoglycan cell wall
o Gram-negative - 2 membranes, peptidoglycan cell wall in
periplasmic space (thinner than in Gram-positive)
genophore bacterial DNA
theta replication occurs in circular dsDNA
virulence genes genes that contribute organism's ability
cause disease
pathogenicity islands group of virulence genes on bacterial
chromosome
virulence plasm ids plasmid containing virulence genes
virulence factors - proteins encoded by virulence genes' '
competence ability of cell to incorporate naked DNA (limited
to exponential phase)
halophiles -live in high salt
Viruses
bacteriophages -low mutation rate, methylated restriction sites
euviruses - high mutation rate, no methylation
nucleocapsid - viral genome together with the capsid
capsid - one or several proteins, arranged in regularly repeating
layers and patterns that encloses viral genome
capsomere - capsid subunit
enveloped viruses- nucleocapsid enclosed by lipid bilayer that virus
acquires in budding from host
Virual reproduction
lysogenic - integration and replication of viral DNA win host
genome
o Provirus / prophage - virus that can intetegrate into host
DNA
o Iysogens - cells containing prophages
lytic replication in cytosol & lysis of celi (or budding off)
type III secretion system acts like syringe that injects toxic proteins
Baltimore classification
1- dsDNA viruses replicates in nucleus
11- ssDNA viruses replicates in nucleus ds intermediate
111- dsRNA viruses replicates in cytoplasm, monocistronic
o Segmented genomes, capsidic
IV & V ssRNA viruses
o 5' cap & 3' tail
o Replicate in cytoplasm
o IV - (+)ssRNA viruses - polycistronic
o V - (-)ssRNA viruses - converted to positive sense by viral
polymerase, monocistronic
VI- ssRNA-retroviruses - reverse transcriptase (2x +ssRNA)
VII - dsDNA-retroviruses - reverse transcriptase
(+) plus strand RNA - functions as mRNA
RNA viruses - often segmented genomes
Complement system
interacting soluble proteins made primarily by liver that
circulate in blood
most inactive until infection
amplify & "complement" action of antibodies
some are also pattern recognition receptors
activation
primary pathway - interaction
alternate pathway - direct stim by cell walls of
microogranisms
Antigen
most large molecules (virtually all proteins & many
polysaccharides)
epitope/antigenic determinant- molecular structure win
antigen recognized by T-cell receptors
hapten -too small to illicit immune response on its own,
must be linked to larger molecule
avidity total binding strength of ali antibodies (polyvalent)
clonal selection theory animal randomly generates vast diversity of
lymphocytes, activates lymphocytes that react against antigens
primary antibody repertoire different antibodies capable of
.. production prior to any antigen stimulation (preimmune)
secondary antibody repertoire - increased diversity of Ig classes &
antigen-binding sites subsequent to antigen stimulation
Innate immune system
dendritic cells
-most efficient innate immune response to PAMPs (pathogen-
associated molecular patterns)
-lots of pattern recognition receptors (TLRs, etc.)
-present microbial antigens to T cells in peripheral lymphoid
organs
natural killer cells (NK cells) - induce apoptosis in virus-infected
cells
- recognize class I M HC proteins
"professional phagocytes"
-macrophages (mature monocytes) - ingest large particles and
cells
-neutrophi/s - can eject chromatin to trap bacteria
o Formylmethionine-peptides very potent chemoattractants
for neutrophils
basophi/s - secrete histamine
eosinophils destroy parasites & modulate inflammatory
responses
lymphoid organs - thymus (central/primary), lymph nodes, spleen, &
appendix
Stages of lymphocyte maturation
naiVe cells - encounter their antigen for first time
effector cells - stim'd naIve cells that carry out immune response
memory cells - more easily become effector cells in later encounters
wi same antigen
Effector cells
very little ER (do not secrete antibodies)
secrete cytokine interferon-g (lFNg), greatly enhances anti-viral
responses.
o cytokine - 4 a helices
o blocks viral replication
o increases expression of MHC genes
T Cells - mature in thymus
helper T cells
activate cytotoxic T cells to kill infected target cells
stim dendritic celis to remain active
THl cells
secrete cytokines interferon-g (lFNg) & tumor necrosis factor- a
(TNFa)
o increase monocyte & neutrophil production in bone
marrow
o activate endothelial cells - cause monocytes & neutrophils
in the blood to adhere
o chemokines - positively charged cytokine that directs
migration of monocytes & neutrophils to infection
. TH2 cells
secrete cytokines interleukins 4 and 10 (lL4 and ILlO)
o stim B cells to make antibodies (lgM, IgA, IgE, & some IgG)
cytotoxic T Cells
induces apoptosis in infected cells
o Fas ligand - binds receptor initiating caspase
cascade
per/orin - pore-forming protein by cytotoxic T cens
cross-presentation - activation by uninfected dendritic cells
regulatory T cells - suppress activity of other T celis
B Cells
large role in humoral immune system
make receptor & secretory antibodies (expanded ERs)
plasma cells
memory B cells - form following primary infection
megakaryocytes
o responsible for platelets
o extraordinarily large wi highly polyploid nucleus,
remains in bone marrow
Immune Receptors
T Cell Receptors (TCRs) - antibody-like heterodimers
immunological synapse - interface blw antigen-presenting
cell & lymphocyte
diversity thru D-to-D joining, junctional imprecision, & N-
nucleotide addition
Toll-like receptor (TLR) - pattern recognition receptors that triggers
innate immune response to pathogens (microbial DNA, etc.)
NOD proteins - exclusively intracellular pattern recognition
receptors
MHC (major histocompatibility complex)
class I MHC - on all cells, display peptides from cytosolic
proteins
o a chain & (CD8)
o usually bind to cytotoxic Tcelis
class II MHC - macrophages, dendritic & B celis, display
endocytosed peptides
o usually bind to helper & regulatory T cells (CD4)
Antibodies/immunoglobulins (Ig) - 2 heavy & 2 (K & A) light chains,
Fc tail & Fab binding site
Primary classes
IgM - Il heavy chain - monomer or pentamer; first & major
antibody in primary immune response, activates complement
IgD - <5 heavy chain - monomer; second class to appear on
surface of B celis
Secondary classes
IgA - a heavy chain - monomer, dimer or trimer; principle class
in secretions (saliva, tears, milk)
IgE - E heavy chain - monomer; allergic response, binds mast
cells in tissues & basophils in blood (allergic reactions)
IgG - Y heavy chain - monomer; major antibody in secondary
immune response, most abundant in blood; activates
complement
o tail binds to Fc receptors on tnacrophages & neutrophils
class switching - after stimulation, B cells switch from IgM & IgD to
other antibodies (does not involve light chains)
affinity maturation - antibody affinity for antigen increases over time
polyclonal antibodies - produced by many different B cells
responding to one antigen that bind to different epitopes
monoclonal antibodies - identical antibody produced by identical B
cell in response to one antigen
V(D)J recombination
light-chain V region (V + J -7 VJ)
-long V gene segment
- short J gene segment (joining)
heavy"chain V region (0 + J -7 OJ + V -7 VDJ)
-0 gene segment (diversity)
. " - short J gene segment (joining)
-long V gene segment
RSS (recombination signal sequences) - before Js & after Vs
RAG (recombination activating genes)
-cuts bin gene segments & recombination signal sequences
- recruits rejoining enzymes (DNA double-strand repair)
junctional diversification - random loss and gain of nucleotides at
joining sites of antibody gene segments
allelic exclusion - choice between maternal & paternal chromosomal
loci
activation-induced deaminase (AID) - deaminates C to U in V region
producing mismatches, repair produces mutations
somatic hypermutation - accelerated mutation rate in B celis
Self-tolerance
central tolerance
receptor editing self-reactive lymphocytes change their
antigen receptors
clonal deletion, self-reactive lymphocytes apoptose
peripheral tolerance
clonal inactivation (clonal anergy), self-reactive lymphocytes
functionally inactivate
clonal suppression regulatory T cells suppress self-reactive
lymphocytes
METHODS
DNA libraries
genomic DNA library- collection of plasmids containing genome of
organism (not necessarily in whole genes)
cDNA library- collection of plasmids containing cDNA only (DNA
complementary to mRNA), not full genome
o contains info on level of varying expression - some cDNA
present in duplicit
o contains uninterrupted coding sequences
Mutation methods
Site-directed mutagenesis - DNA is cut, DI\IA is inserted
Oligonucleotide-directed mutagenesis - complementary segment
made wi single base-pair mismatch
hybridization & replication result in site mutation
induced somatic mutations - modification of DNA in subset of celis
at late stage of development
genetic mosaic- organism subjected to induced somatic
recombination
DN,A fingerprinting - does not normally use DNA microarrays
number of tandem copies of a simple sequence repeat
uses PCR, restriction endonuclease digestion & southern
blotting
RFLP - restriction fragment length polymorphism
Lasers
laser capture microdissection
optical tweezers - use of laser to move organelles, chromosomes
Microscopy "
deconvolution - images from a series of focal planes are combined to
present an in focus plane
Light (up to .2 11m)
Techniques
confocal- excludes out of focus light
o multiphoton confocal- uses multiple photon absorption
for greater penetration
fluorescence microscope - uses fluorescent dyes
",. TlRF (total internal reflection fluorescence) - use of indirect
light to image cell- visualization of single molecules
Electron
Types
Transmission (TEM) (up to Inm (10 angstroms) for biological
specimens) [0.1 nm for non-biological]
o difficult preparation/fixing - fixed, dehydrated, resin-
embedded
o supercooled into vitreous ice (noncrystalline "glass")
Scanning (SEM) (up to 10nm res)
o 3D structure of surface (10nm res) - used for cells & tissues
not subcellular components
Techniques
immunogold - gold particles attached to antibodies
metal shadowing - heavy metal evaporated onto specimen
from oblique angle
o negative staining - additional step to invert image
crystallography - EM + electron diffraction analysis
cryoelectron - specimen can be viewed w/o fixation, staining,
drying (cryogenic container at -160C)
o cryostat - cuts thin slices of specimen
single-particle reconstruction - thousands of images of
identical molecules are arranged together
tomography- 3D through rotation of specimen in TEM and
combination of images
atomic force - allows manipulation of individual molecules w/
silcon probe
Mass spectrometry
precise determination of the mass of a protein (mass used to
ID unknown protein)
determine length of hydrocarbon chains or position of double
bonds
Techniques
Matrix-assisted laser desorption ionization (MALOI) - soft
ionization process used on biomolecules
Time of Flight (TOFMS) - mass-charge ratio of ions used to ID
molecules
TOF/TOF - tandem mass spectrometer - change in mass of IDs
amino acid lost (leucine/isoleucine same mass)
Restriction enzymes (endonucleases)
Type I - cleave DNA at random sites (up to 1000 bp from
recognition site)
Type 11- cleave DNA within known recognition sequence
Type 111- cleave ~ 5 b p from recognition site (requires ATP)
isoschizomers - restriction enzyumes from different species w/
same recognition sequence
peR (polymerase chain reaction)
single-stranded DNA primers
deoxynucleotide triphosphates (dNTPs)
dideoxynucleotide triphosphates (ddNTPs)
stringency-level of homology; fidelity & specificity of hybrid
annealing
fidelity - depends on temp, pH, [NaCI], & probe sequence
U:;R (ligase chain reaction) - good for detection of alleles, point
mutations
Vectors
plasmids 5,000-400,000 bp - difficult to clone segments longer
than 15,000 bp, mUltiple per cell
o cosmid - have euk & prok initiation sequences
phages 40,000-53,000 bp - ID by plaque formation
BACs (Bacterial Artificial Chromosomes) - plasm ids for cloning
DNA of 100,000-300,000 bp
o Accommodates largest inserts (up to 300k bp)
o from F plasmid, 300,000 - Imil bp, 1-2 copes per
YACs (Yeast Artificial Chromosomes) -linear, telomere stable if
longer than 150,000 bp
o contain features necessary for propagation as an
independent chromosome (centromere, telmomeres, ori)
o Plasm ids for yeast transformation - up to 2,000,000 bp
o Not recircularized, remain as linear molecules
Shuttle vectors - plasm ids that can be used to transform
different species
Expression vectors - cloning vector w/ transcription &
translation signals needed for regulated expression
o plasmids designed to produce large amounts of mRNA
o contains promoter, ribosome-binding site, & transcription
termination sequence
Viral vectors - used in animals for transformation
Delivery (of non-permeable substances - vectors, etc)
micropipette injection - injection of DNA directly into cell
and/or nucleus
electroporation - electric shock leaves bacterial cell membrane
temporarily permeable to DNA uptake
membrane-enclosed vesicles
gold particles - shot into cells (DNA coated)
Fluorescence
GFP (Green Fluorescence Protein)
FRET (fluorescence resonance energy transfer)
cellular location of reactions
FACS (Fluorescent-activated cell sorting) - used to separate cells by
DNA content, size, surface markers
FRAP (fluorescence recovery after photobleaching) -laser beam
extinguishes fluorescence --recovery recorded
Electrophoresis
SDS (sodium dodecylsulfate) - determines purity and
molecular weights of proteins
o SDS treated proteins - smaller weight travels faster
o anomalous if protein contains carbohydrates in large %
isoelectric focusing - organic acids and bases establish pH
gradient across gel, target protein migrates to pH pi
two-dimensional use of both SDS& isoelectric focusing
pu Ised field gel electrophoresis - alternating voltage allows for
better resolution of large DNA molecules (>15-20kbs)
gel-mobility shift assay - detects DNA bound proteins through
retardation of movement
denaturing polyarylamide - used in DNA sequencing
Blotting - electrophoresis; probing & hybridizaton in RNA/DNA,
antibody affinity in proteins \'
allows detection of minor sample component and provides
estimated weight
target sequence usually twice as long as probe
o southern - DNA
o northern - RNA
o western (immunoblotting) - protein
Column chromatography
reservoir - container
stationary phase column
mobile phase - solution to be run through column
effluent - solution passed thru column
Types
cation-exchange - negatively charged stationary phase
hydrophobic hydrophobicity
gel filtration / size-exclusion - separates according to size
(smaller proteins slowed down by pores/cavities)
affinity - functional groups in stationary phase bind to proteins
w/ varying affinity
HPLC (high performance liquid) - high pressure
chromatography that limits diffusional spreading maximizing
resolution
absorption or thin-layer - separates lipids of diff polarity
Antibody techniques
effective for detecting & quantifying hormones (can detect
very small amounts of specific material)
Techniques
RIA (Radio Immuno Assay) quantifies amount of hormone in
sample
o incubate sample w/ hormone-specific antibody & purified
radiolabeled hormone
o unlabeled hormone competes & displaces radiolabeled
hormone from antibody
ELISA (enzyme-linked immunosorbent assay) - screens for
presence of an antigen
Protein micro array / chip array of antibodies to ID proteins
Coimmunoprecipitation precipitation of complexed proteins
w/ targeted ones
epitope tag - use of known epitope to tag protein
Fused Cells
hetercaryon fused cells w/ separate nuclei
hybridomas - fused B lymphocyte w/ transformed (tumor line cell) B
lymphocyte - molecular factory for monoclonal antibodies
DNA Sequencing
Sanger / dideoxy method - ddNTPs terminate DNA synth, creating
strands of various lengths sequenced thru electrophoresis
Maxam-Gilbert- based on chemical modifications of DNA
Edman degradation label & removal of 1 AA at time (res: ~ 5 AAs)
sequenator- automated Edman degradation
chromosome walking - sequencing thru cyclic creation of primers
Protein Sequencing
TOF/TOF - tandem mass spectrometer - change in mass of IDs
amino acid lost (leucine/isoleucine same mass)
Sequence evolution (species relatedness)
Blosum62 (62% identical) - blocks substitution matrix, mathematical
analysis of genome evolution
Protein interactions
.Surface plasma resonance (SPR) detects binding interactions by
ch,ange in resonance angle, good for timing of interactions
yeast two-hybrid system
IDs protein interactions (w/ known "bait"), co-activators & co-
repressors
reporter genes easily assayed enzymes
Protein conformation
NMR spectroscopy - protein conformation
Genetic Analysis
genetic screen - genetic analysis of numerous individuals to ID
mutant allele responsible for mutant phenotype
pleiotropy - single gene influences multiple phenotypic traits
epistasis analysis - analysis of combinations of mutations in orderto
determine order of gene action (epistatic - occurring prior)
complementation test - tests if 2 mutations are products of
mutations in one or two genes
Cre-Lox Recombination - gene knockout: lox sites - target gene, cre
recombinase remove it
protein synthesis - ~ 1 AAs - on polymer support
Gene Expression
DNA microarrays - rapid + simultaneous screening of thousands of
genes wIn species
probed w/ mRNA or cDNA ID what genes are being expressed
Comparative genomic hybridization (CGH) - DNA microarray
comparison to see what genes over/under expressed
DISORDERS, TOXINS, REAGENTS, &
DRUGS
Disorders
Huntington's disease - autosomal dominant disorder
Parkinson's disease - underproduction of dopamine
maple syrup urine disease lack of debranching AA deg. Enzyme
abnormal brain development & infancy death
anemia - hemoglobin deficiency
beriberi -lack of Vitamin B1 (thiamine) - involved in breakdown of
glucose
hypoxin -lowered oxygenation of peripheral tissues
Jaundice accumulation of bilirubin in blood b/c not enough
glucuronyl bilirubin transferase to process to bilirubin diglucuronide
diabetes - failure of insulin secretion or action, fail to synth fatty
acids from carbohydrates or amino acids
acidosis & ketosis lowered blood pH caused by ketone bodies
(acetoacetate D-j3-hydroxybutyrate) in blood
Disorders
Huntington's disease - autosomal dominant disorder
Parkinson's disease - underproduction of dopamine
Anemia - hemoglobin deficiency
Beriberi -lack of Vitamin B1 (thiamine) - involved in breakdown of glucose
hypoxin -lowered oxygenation of peripheral tissues
Jaundice - accumulation of bilirubin in blood blc not enough glucuronyl bilirubin transferase to process to bilirubin diglucuronide
diabetes - failure of insulin secretion or action, fail to synth fatty acids from carbohydrates or amino acids
acidosis & ketosis -lowered blood pH caused by ketone bodies (acetoacetate in blood
Toxins
a-amanitin - (death caps) inhibits RNA polymerase
"
colchicine - inhibit MT assembly by capping ends(blocks cell division), induces polyploidy in plants
cyanide, CO, azide - blocks electron transport from Complex IV (cytochrome oxidase) to O
2
- blocks elongation "
dinitrophenol (DNP) - dissipates proton gradient in mitochondria, eliminating ATP synthesis
flouroacetate - inhibits CAC
diphtheria toxin - inactivates EF-2, blocking elongation
phalloidin - (death caps) prevent actin disassembly
excess triacylglycerol (TAG) in skeletal muscle & liver can be toxic
Drugs
allopurinol- purine analog that blocks purine breakdown, lowering uric acid levels
anti-inflamatory steroids
aspirin (NSAlDs) - inhibits cyclooxygenase (COX), essential for arachidonate conversion to prostaglandins & thromboxanes
prednisone - inhibits arachidonic acid release by phospholipase A2 blocking prostaglandin formation
cardiac steroids (digitalis, oubain) - inhibit Na+,K+-ATPase (l'[Na+], l'[Ca
2
+]), increase contractile force
Antibiotics
actinomycin - prevents elongation, inhibiting transcription
antimycin A - inhibits oxidation of CoQ by binding Complex III (cytochrome bCl complex)
erthyromycin - binds 50S subunit, preventing translocation
oligomycin - blocks Fa (proton pore) of ATP synthase
puromycin - causes premature release of protein chains, inhibiting synthesis
streptomycin - binds 30s subunit, preventing initiation
penicillin - prevents synth of peptidoglycan cross-links in cell wall of gram-positive bacteria
fusidic acid - prevents release of EF-G-GTP, blocking tranlocation
chloramphenicol- inhibits peptidal transferase activity of bacterial ribosomes
tetracycline - binds 16S rRNA, blocking aminoacyl-tRNA
Reagents
mercaptoethanol & performic acid - oxidizes disulfide bonds
dansyl chloride - used in n-terminal AA analysis
cyanogens bromide - used to cleave peptide chains at methionyl residues
iodoacetate/iodoacetamide - used to modify cysteine side chains into thioethers, preventing disulfide bond reformation
phenol & chloroform - precipitate proteins out of lysate
Mutagens
Intercalating agents
o Planar molecules that lodge blw stacked DNA bases causing DNA pol to insert or skip bases
o proflavin, acridine organe, & ethidium bromide
base analogs
o less stable, causes mismatches
o 5-bromouracil & 2-aminopurine
alkylating agents - cause mismatch by chemically modifying bases
o hydroxylamine - GC --7 AT
spermatogonium -(rep.)--7 prim. spermatocyte -(mei-I)--7 sec. spermatocyte -(mei-II)--7 spermatid - spermatozoa (mature sperm)
oogonium -(rep.)--7 prim. oocyte -(meiosis 1)--7 sec. oocyte -(mei-II)--7 oovid - ovum (mature egg)
pyridoxal 5-phosphate (PLP) - Schiff base + E-amino
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