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com






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Copyright 2005-2007 Ayaz Najafov www.GREBIO.com


TABLE OF CONTENTS
(Books that were used to prepare this summary eBook)


Biochemistry
by Jeremy M. Berg, John L. Tymoczko, Lubert Stryer
- 2006, 6
th
edition
- ISBN: 0716787245
- 1040 pages
- Publisher: W. H. Freeman
- Price at Amazon.com: 160 $
- Link: http://www.amazon.com/gp/product/0716787245?ie=UTF8&tag=neyhaber-
20&linkCode=as2&camp=1789&creative=9325&creativeASIN=0716787245



Molecular Biology of the Cell
by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter
- 2002, 4th edition
- ISBN: 0815332181
- 1616 pages
- Publisher: Garland Science
- Price at Amazon.com: 104 $
- Link: http://www.amazon.com/gp/product/0815332181?ie=UTF8&tag=neyhaber-
20&linkCode=as2&camp=1789&creative=9325&creativeASIN=0815332181



Genetics
by Robert F. & Hedrick, Phillip W. Weaver
- 1997, 3rd edition
- ISBN: 0697100219
- 672 pages
- Publisher: William C Brown Pub
- Unavailable from Amazon.com



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Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 1: Prelude

Limit of resolution of light microscope = 200nm
Diameter of DNA = 20A
o

A typical covalent bond in a biological molecule has energy of 15-170 kcal/mole.
Average thermal energy at the body temperature is 0.6kcal/mole. Therefore, even an
unusually energetic collision will leave a covalent bond intact.

There are 4 types of major non-covalent bonds or intermolecular interactions:

1) Electrostatic interactions (also known as ionic bonds, ion pair, salt bridge and salt
linkage):
Coulombs law governs these interactions
Strongest in vacuum (Diaelectric constant = 1)
Weakest in water (Diaelectric constant = 80)
E.g. Na(+).Cl(-)
Charged groups are required

2) Hydrogen bonds:
H atom is shared by two other atoms such as N and O
H is more tightly linked to H-bond donor and less tightly linked to H-bond acceptor.
3-7 kcal/mol
both charged and uncharged groups can form H-bonds

3) Hydrophobic:
non-specific
between two hydrophobic regions of a molecule

4) van der Waals bonds:
non-specific
distance = 3-4A
o

1 kcal/mol



covalent > electrostatic > hydrogen > hydrophobic > van der Waals





I: ionic strength
C: concentration (Molars)
Z: charge

e.g. Ionic strength of a 0.5M MgCl
2
is:

MgCl
2
Mg
++
+ 2Cl
-
0.5M 0.5M 1M

I = (0.5 2
2
+ 1 1
2
) = (2 + 1) = 1.5
I = C
i
Z
2
i

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HA H
+
+ A
-




pH of a solution is a measure of the H
+
concentration of that solution.
pKa is a pH value at which an acid is half dissociated, i.e. when [A
-
] = [HA].



Henderson-Hasselbalch Equation:



Free energy change (G) must be negative for a reaction to be spontaneous.

Hydrophobic effect: nonpolar molecules tend to aggregate in water because the entropy
of water is increased through the release of water molecules.

Fischer projection:
Horizontal lines out of the page, towards the viewer
Vertical lines into the page, away from the viewer






Chapter 2: Biochemical Evolution

Ribozymes: catalytically active RNA molecules

DNA vs. RNA at stability:
DNA is 100 times more stable than RNA at neutral conditions.
The 2-OH group of RNA makes it more susceptible to base-catalyzed hydrolysis.
A CH
3
(methyl) group of Thymine that is absent in Uracil, facilitates DNA repair.

Amphipathic molecules: molecules that contain both hydrophilic (water-loving) and
hydrophobic (water-avoiding) groups (e.g. phospholipids)

Osmosis: movement of solvent (water) across a membrane, in an attempt to equalize the
concentrations of solutes on both sides of the membrane.

] [
] [
log
HA
A
pK pH
a

+ =
] [
] ][ [
HA
A H
K
a
+
=
a a
K pK log = ] log[
+
= H pH
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 3: Protein Structure and Function

Zwitterions: dipolar ions with both a negative and a positive group (e.g. amino acids)

Functions of proteins
1) Enzymatic catalysis
2) Transport and storage
3) Coordinated motion
4) Mechanical support
5) Immune protection
6) Generation and transmittance of nerve impulses
7) Control of growth and differentiation



Average molecular weight of amino acids = 110 g/mol = 110 daltons = 0.11 kDa.
Only L-amino acids are constituents of proteins (not D-amino acids).
Only Glycine is optically inactive.
Peptide bond is rigid and planar.
Almost all peptide bonds in proteins are trans.
11 out of 20 amino acid side chains can participate in H-bonding.
Polypeptide chains fold in such a way that its hydrophobic chains are buried inside.


Disulfide bonds:
Formed between two Cysteine residues on the same or different polypeptide chains.
Can be reversibly cleaved with reducing agents: -mercaptoethanol or Dihiothreitol
(DTT)


Aromatic
(FWY)
Phenylalanine
Tryptophan
Tyrosine
Sulfur-containing
(MC)
Methionine
Cysteine
Acidic
(DE)
Aspartate
Glutamate

Basic
(KRH)
Lysine
Arginine
Histidine
Phosphorylatable
(STY)
Serine
Tyrosine
Threonine

Primary structure: amino acid chain
Secondary structure: alpha-helix, beta-sheet, turns, loops
Tertiary structure: compact structures with nonpolar cores (for water-soluble proteins
Quaternary structure: several subunits assemble into a multisubunit complex


Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 4: Exploring Proteins

Each centrifugation step yields two fractions: supernatant and pellet.

Differential centrifugation: Different cellular organelles have different densities and
therefore will sediment at different centrifugation conditions (centrifugation field strength
in g and length of the centrifugation).

Lysed cells can be separated into three distinct fractions by using differential
centrifugation:

1) 500g for 10 minutes pellet: nuclear fraction
2) 10,000g for 20 minutes pellet: mitochondrial fraction
3) 100,000g for 60 minutes pellet: microsomal fraction (ER, Golgi, etc)

Supernatant of the last step will contain the soluble cytosolic proteins.

Salting out: Different proteins have different solubilities and therefore will fall out of
solution and precipitate at different salt concentrations.

Dialysis: Different molecules (e.g. proteins vs. salts) have different sizes and therefore
can be separated by dialysis through semipermeable membranes with pores that allow
passage of small molecules (salts) but not the molecules of interest (proteins).

Gel-filtration chromatography: Different proteins have different sizes and therefore can
be separated by using a matrix (insoluble polymer beads 0.1mm in diameter with tiny
pores in them) that allow bigger proteins to spend less time inside the matrix (inside the
pores) and therefore to migrate faster, than small proteins, through a column of such
matrix.

Ion-exchange chromatography: Different proteins have different net charges and
therefore will bind with different affinities to positively/negatively charged matrices.
Proteins bound to such a matrix can be differentially eluted with high concentration salt
solutions.

Affinity chromatography: Different proteins have different specific affinities (for
example protein concavalin A binds specifically to glucose) for different molecules and
therefore a matrix that has those molecules covalently attached to it can be used to
differentially separate certain proteins from other proteins.

High Pressure Liquid Chromatography (HPLC): All the chromatography techniques
described above can be much more efficient if HPLC is employed. The columns used in
HPLC have a greater resolution power because the matrix units are much finer (imagine
that the beads are much smaller) and thus have a greater number of sites for
interaction between the matrix and the proteins. Since the matrix is very fine, high
pressure is used in the chromatography columns to get fast flow rates. The important
properties of HPLC are 1) much higher resolution power and 2) much faster flow rates,
when compared to regular chromatography techniques.

Gel electrophoresis: If proteins are denatured with a detergent SDS (Sodium Dodecyl
Sulfate) and the SS (disulfide) bonds are cleaved with a reducing agent such as -
mercaptoethanol or Dithiothreitol (DTT), proteins will assume a linear structure (primary
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structure) with SDS anions bind to the denatured polypeptide chain at a ratio of about 1
SDS anion per 2 amino acid residues. In this shape, protein migration in a sieving matrix
such as polyacrylamide gel is almost always linearly proportional to logarithm of the
molecular weight of the protein. Small proteins will migrate faster than larger proteins in
such a matrix because the matrix (the gels molecular sieving structure) retards small
molecules less than it retards large molecules. Note that the gel electrophoresis described
here is SDS-PAGE, but there is also a non-denaturing version of gel electrophoresis, which
is not that much widely used and which does not separate proteins based on their sizes
it separates proteins based on their hydrodynamic volumes.

Note: Pay attention to the fact that the gel-filtration chromatographys logic is quite the
opposite of gel electrophoresis logic: in gel-filtration chromatography, small proteins
spend more time in the matrix and thus migrate slower than the large proteins. In gel
electrophoresis, small molecules are retarded less by the matrix than large proteins are,
and thus migrate faster.

Isoelectric focusing: Different proteins have different net charges and therefore will
have a net charge of zero at different pHs (pI value: pH at which the net charge is totally
neutralized by the ions of the solution).Thus, different proteins can be immobilized
(focused) in a gel that has a pH gradient in it. Such pH gradients are achieved by using
ampholytes.

Two-dimensional (2-D) electrophoresis: Combination of isoelectric focusing
(separation based on pI) and SDS-PAGE (separation based on size) gives a new gel-based
technique that allows to further separate different protein from each other. The total
resolution power of 2-D electrophoresis is much greater than resolution powers of
isoelectric focusing and gel electrophoresis.




Carbohydrate rich proteins and membrane proteins migrate anomalously on SDS-PAGE
gels: they do not obey the relationship migration is linearly proportional to log(MW).
0.1g of protein can be detected by Coomassie Blue.
0.02g of protein can be detected by silver staining.
Isolecetric point (pI) is the pH at which the net charge of the protein is zero.
BSA (acidic protein): pI = 4.8; Cytochrome C (basic protein) : pI = 10.6
The dependence of protein solubility on salt concentration salting out. (NH
4
)
2
SO
4
is
the most commonly used salt.
CNBr (Cyanogen bromide) splits the polypeptide chains only at the carboxyl side of the
Methionine residues.
The resolution of fluorescent microscopy is 200nm.
Immunoelectron microscopy can resolve 10nm or finer.
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 5: DNA, RNA, and the Flow of Genetic
Information

Hypochromism: dsDNA (double-stranded DNA) absorbs less UV light than a ssDNA
(single-stranded DNA) molecule. In other words, a denatured DNA molecule absorbs more
UV light than a non-denatured one.

DNA level (transcription initiation):
Prokaryotic promoter: -35 region + Pribnow Box
Eukaryotic promoter: CAAT Box (sometimes present) + TATA (Hogness) Box

RNA polymerase, transcription factors, silencers and enhancers and related proteins all
play around promoter before the initiation of transcription. Once the transcription initiated,
RNA polymerase does not need any of these initiation factors.

RNA level (translation initiation):
Prokaryotic translational start signal: Purine-rich sequence (Shine-Dalgarno) + AUG
Eukaryotic translational start signal: 5-CAP + AUG




Viruses may have their genome in the form of RNA or DNA.
DNA replication is semiconservative, semidiscontinuous and usually bidirectional.
The plane of the bases is perpendicular to the DNA helix axis.
The diameter of DNA helix is 2nm.
Adjacent bases are separated by 3.4A
o
and 36 degrees rotation. Hence, helical
structure repeats after each 10 bases on each chain = 34A
o
.
Tm (melting temperature) is defined as the temperature at which half of the helical
structure is lost.
X174 phage infects E. coli and has ssDNA as its genome.
Nucleotides in DNA and RNA are linked by 35 phosphodiester bonds.
dsRNA is like A-DNA in structure.
Supercoiled circular DNA is more compact and migrates faster in gels than relaxed
DNA.
Many exons encode separate protein domains.
Introns usually start with GU and end with AG with a preceding pyrimidine-rich
sequence.
Shine-Dalgarno sequences base-pair with ribosomal RNA (16S).

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 6: Exploring Genes

The Basic Tools of Gene Exploration:

Restriction Enzyme Digestion Analysis (REDA):

Restriction enzymes = endonucleases
Isolated from bacteria
Have a protective role - to restrict phage DNA from entering the bacterial genome.
Recognize specific 4-8bp long DNA sequences
The recognition sequences are usually palindromes (see below)
Cut both DNA strands within or near those recognition sequences.

5-protruding blunt


Palindromes: words or sequences that can be read from beginning to the end and from
the end to the beginning and give the same meaning. E.g. RADAR.

E.g. for DNA: GAATTC will be read GAATTC if it read from 5 to 3 on both strands.
5-GAATTC-3
3-CTTAAG-5

There are 3 types of ends that form upon digestion with restriction enzymes:
1) 5-protruding (sticky)
2) 3- protruding (sticky)
3) Blunt end

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It is easier to ligate two sticky ends than two blunt ends.
A blunt end and a sticky end cannot be ligated.
Blunt ends can be ligated with T4 DNA ligase but not with other DNA ligases.
Blunt ends can be ligated with PCR products that were generated by T4 DNA
polymerase, Pfu or Klenow (but not Taq).
Sticky ends can be filled in with T4 DNA polymerase and then ligated with a PCR
product (that was generated by T4 DNA polymerase, Pfu or Klenow) or a blunt-
ended DNA fragment.
Polynucleotide kinase adds P
i
to 5 and removes P
i
from 3
Plasmids: accessory circular DNA molecules with certain genes, like antibiotic-
resistance genes, that give advantages to the host bacteria that carry them.
Unlike RNA, DNA is resistant to alkaline hydrolysis.
S1 nuclease recognizes unpaired nucleotides (used to remove loops)
T4 phage ligase can ligate blunt ended double helices.



Blotting Techniques

Botting techniques are usually involve transfer of macromolecules (DNA, RNA or proteins)
onto an absorptive sheets (membranes) made of nitrocellulose or PVDF (Polyvinylidene
Difluoride) and highly-specific detection of the transferred molecules. In most cases, an
electrophoretic step precedes the blotting step, which helps to resolve the mixtures of
molecules and detection of a single type of molecule at an expected molecular size.

There are 3 major blotting techniques:

Western Blotting
- transferred molecule type: proteins
- specificity reagents: mono- or polyclonal antibodies (IgG or IgM) that detect a single
type of protein molecule.
- detection reagents: a secondary antibody that detects the primary antibody (specificity
reagent) and carries an enzyme (alkaline phosphatase or horse radish peroxidase) that is
used to yield chromogenic (insoluble colored product) or chemiluminescent signal.

Monoclonal antibodies: a single antibody type that detects a single site (epitope) on the
target protein. Produced by using hybridomas. Usually are generated in mice.

Polyclonal antibodies: a mixture of all antibodies that detect the target protein (each
antibody type, i.e. each monoclonal in the polyclonal mixture, will presumably bind a
different epitope on the target). Produced by injecting purified target protein or chemically
synthesized peptide fragment of it. Usually are generated in rabbits or goats.

Northern and Southern Blotting
- transferred molecule type: RNA (Northern) and DNA (Southern)
- specificity reagents: DNA or RNA probes (up to 300 nucleotides) that detect a single type
of DNA or RNA molecule.
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- detection reagents: the probes are usually labeled with either a radioisotope, biotin or
digoxigenin (DIG). A secondary antibody that detects biotin or DIG and carries an enzyme
(alkaline phosphatase or horse radish peroxidase) that is used to yield chromogenic
(insoluble colored product) or chemiluminescent signal. If the probe is radio-abeled,
autoradiography method is used to detect the signal.


DNA Sequencing

There are two common DNA sequencing methods:
1. Maxam-Gilbert method (cleavage of
32
P labeled DNA molecules)
2. Sanger method (dideoxy termination)

Synthesis of nucleic acids
Polymerase Chain Reaction (PCR)

Polynucleotide phosphorylase:
- Template independent
- Uses dNDP as substrates
(RNA)
n
+ dNDP (RNA)
n+1
+ Pi

Klenow fragment:
- Derivative of DNA polymerase I
- Lacks N-terminal part of the polymerase lacks 53 exonuclease activity
- Plus, has two missense mutations that abolish its 35 exonuclease activity
- Used for labeling probes and primer extension method
- Used for filling in or digesting out 3-overhangs (sticky ends) to generate blunt ends
- Used for synthesis of dsDNA from ssDNA




DNA ligase requires:
1) 3-OH
2) 5-PO
4

3) double helix form
4) energy: ATP or NAD
+


Insertional inactivation:
1) antibiotic resistance
2) -galactosidase gene (blue/white screening technique)

-Phage:
1. ~15KB inserts.
2. Too small or too big inserts cannot be packaged.
3. Enter bacteria much more easily than plasmids.
4. ~100 progeny per generation of lytic pathway.

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M13 phage:

Filamentous virus
Enters E. coli through the sex pilus
Only (+)ve strand is packaged into the virus
~1000 progeny per generation
Does not kill the host! Large quantities can be harvested
Universal sequencing primer can be employed
Ideal for sequencing but not for long term propagation of DNA: inserts >1KB are
not stably maintained.

Chapter 7 ("Exploring Evolution") was completely omitted due to low or no relevancy t the GRE Subject
Biochemistry, Cell and Molecular Biology test.

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 8: Enzymes: Basic Concepts and Kinetics
and
Chapter 10: Regulatory Strategies: Enzymes and
Hemoglobin


Almost all known enzymes are proteins but there are RNA enzymes too (ribozymes).
Enzymes are highly specific and they increase the reaction rates (make them up to
10
6
times faster) (catalysis).
The high specificity of enzymes is achieved by high precision of enzyme-substrate
interaction that is a result of intricate 3D structure of the enzyme.
Catalysis is achieved by stabilizing the reactions transition states (the highest-energy
species).
Many enzymes require cofactors.
Cofactors are divided into two classes: metal ions like Mg
++
or Zn
++
and coenzymes -
small organic molecules like PLP and vitamin-derivatives.
Apoenzyme + cofactor = holoenzyme.
Allosteric interactions: interactions between spatially distinct sites of a protein.

Myoglobin and -chain of hemoglobin are virtually superimposable. However, their amino
acid identity is only 17% Quite different amino acid sequences can lead to very similar
3D structures.

Unlike myoglobin, hemoglobin is an allosteric protein:
1) O
2
binds to hemoglobin in a sigmoidal fashion and to myoglobin in a hyperbolic.
2) O
2
binding to hemoglobin is CO
2
and pH dependent (unlike to myoglobin).
3) BPG regulation is present only in hemoglobin
4) Myoglobin has higher affinity (lower Kd) than hemoglobin.

Saturation Y = fractional occupance of all the binding sites in a solution
(ranges from 0 to 1)

Affinity is expressed in terms of K
d
(P
50
) = concentration of the ligand at which 50% of all
the sites in a solution are occupied, i.e. when Y = 0.5

A + B AB




K
d
affinity

Hills plot:
Hills plot is defined as log(Y/1-Y) vs. [B]
Y (fraction bound) / 1-Y (fraction unbound) = ([B]/K
d
)
n
Log(
Y
Y
1
) = n log[B] n log (K
d
)
When n = 1 NO Cooperativity (as in myoglobin)
If the binding is cooperative A + nB A(B)
n
(where n is the Hill coefficient)
Then, Y becomes:

] [
] ][ [
AB
B A
K
d
=
] [ ] [
] [
] [ ] [
] [
d
K B
B
A AB
AB
Y
+
=
+
=
] [ ] [
] [
n
d
n
n
K B
B
Y
+
=
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Cooperativity faster saturation

Bohr effect:
pH

affinity of hemoglobin for O
2

[CO
2
] affinity of hemoglobin for O
2

reciprocally, [O
2
] unloads CO
2
and H
+
from hemoglobin.

BPG decreases the affinity of hemoglobin for O
2
by 26X.
HbF binds BPG less strongly than HbA HbF has higher affinity for O
2
.
BPG binds only to deoxyHb.
BPF stabilizes the deoxyHb quaternary structure by cross-linking the -chains.

OxyHbs heme: planar
DeoxyHbs heme : convex

In an aerobic organism, 0.8 CO
2
are produced per 1 O
2
consumed.
Most of the CO
2
are transported in the blood as HCO
3
-
(bicarbonate):
CO
2
+ O
2
HCO
3
-
+ H
+
(catalyzed by carbonic anhydrase
)

The remainder of the CO
2
is carried by the Hb as carbamate.
0.5H
+
are taken up by the Hb per 1 O
2
released.
deoxyHb has greater affinity for H
+
than oxyHb.

Two models for allosteric mechanism:

1) Sequential (postage stamp analogy):
Assumtions:
a) T&R are the only conformational states accessible to any subunit.
b) T to Rtransition upon ligand binding occurs in each subunit separately.
c) Upon T to R transition, K
d
of a neighbor subunit is affected.

2) Concerted (symmetry is conserved in allosteric transitions)
Assumtions:
a) No TR hybrids: protein is always in T R equilibrium.
b) Ligands bind to T with low affinity and to R in high affinity.
c) Binding of each ligand increases the R over T probability.

Homotropic effects: effects in allosteric interactions due to identical ligand binding
(e.g. O
2
binding to Hb)
Heterotropic effects: effects in allosteric interactions due to different ligand binding
(e.g. CO
2
and H
+
affects binding of O
2
to Hb)

Sickling occurs when [deoxyHb] increases HbS molecules stick.

Regulation of catalytic activity of enzymes:
1) Feedack inhibition (via allosteric interaction)
2) Regulatory proteins (e.g. calmodulin: Ca++ sensing protein)
3) Covalent modifications (e.g. reversible phosphorylation)
4) Proteolytic activation (zymogens: proinsulin insulin, trypsinogen trypsin)



Rate of formation of ES = k1[E][S]
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Rate of breakdown of ES = (k2 + k3)[ES]

K
M
= Michaelis constant





V = velocity of the reaction = moles of product formed per second





V
max
is attained when all the catalytic sites are saturate,
i.e. if [S]>>K
M

M
K S
S
+ ] [
] [
approaches 1 Vmax = k
3
[E
T
]

Therefore, V can be expressed as:





K
M
affinity of E to S when k2>>k3.

Fast enzymes have high K
M
.

Lineweaver-Burk plot is defined as 1/V vs. 1/[S] plot
In this plot,
slope = K
M
/ Vmax
Y-intercept = 1/Vmax
X-intercept = -1/K
M

The equation of this plot is:
1/V = 1/Vmax + K
M
/[S]Vmax

K
M
depends on:
1) Particular substrate
2) pH
3) Temperature
4) Ionic strength

Fraction of catalytic sites filled = f
ES
= V/Vmax

Turnover number = number of S molecules converted into P by an enzyme molecule
when it is fully saturated with the S = k3 (unit: 1/second)
k
cat
= maximal catalytic rate when S is saturating the E.
i.e. V, when [S] is saturating

max max
] [
1 1
V S
K
V V
M
+ = as [S] approaches , V approaches V
max

1
3 2
] [
] ][ [
k
k k
ES
S E
K
M
+
= =
M
T
K S
S E k
V
+
=
] [
] ][ [
3
M
K S
S V
V
+
=
] [
] [
max
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com

k
cat
/K
M
= a criterion in catalytic rate

Kinetic perfection is attained when k


cat
/K
M
is in the range of diffusion limits.

Diffusion limits = 10
8
-10
9
M
-1
s
-1

Enzyme inhibition

a) Irreversible: covalent or non-covalent
b) Reversible:
i. Competitive:
1. inhibition can be overcome by [S]
2. Vmax does not change
ii. Non-competitive
1. inhibition cannot be overcome by [S]
2. Km does not change
iii. Mixed



Allosteric enzymes do not obey Michaelis & Menten kinetics

Transition state analogs:
1. Provide insight into catalytic mechanisms
2. Can serve as potent and specific inhibitors of enzymes
3. Can be used as immunogens to generate catalytic antibodies

Allosteric control
1. Control proteins
2. Reversible covalent modification (e.g. phosphorylation)
3. Proteolytic activation (i.e. zymogens)

Proteins that are entirely extracellular are not regulated by phosphorylation.

Protein Kinase A is activated by cAMP: cAMP binds to regulatory subunit of the enzyme
that has a pseudosubstrate sequence and blocks the catalytic subunit when bound to it.

Thrombin cuts out highly negatively charged parts out of fibrinogen that prevent the
latter from aggregation. Newly formed fibrin molecules aggregate specifically to form a
fibrin clot.

Heparin (anticoagulant) increases the rate of formation of irreversible complexes
between the clotting factors (serine proteases like thrombin) and antithrombin III.
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 9: Catalytic Strategies

Lysozyme is a glycosidase that cleaves the glycosidic bind between NAG (N-acetyl
glucosamine) and NAM (N-acetyl muramic acid).

When NAG oligos are <5, binding still occurs but the hydrolysis is slow.
In fact, (NAG)
3
is a potent inhibitor of lysozyme.

Polysaccharide chains, like polynucleotides (5-3) and polypeptides (N-C), have
directionality: (1-4)
Chitin is a poly-NAG.
Dissociation R-COOH R-COO
-
+ H
+
is less favored in nonpolar medium.
RNase A is a digestive enzyme secreted by pancreas.
RNase A is cleaved by subtilisin to form RNaseS
RNases cut before pyrimidines.
ssDNA binds to RNAases but cant be cleaved.
Phosphonate = non-hydrolyzable analog of esters.
Scissile bind = the cleaved bond (e.g. phosphodiester bond is cleaved by RNases)

Carboxypeptidease A is a zinc protease that hydrolyzes C-terminal bulky amino acids.

Chymotrypsin is a serine protease that cuts after Tyr, Trp, Phe, Met.
Like many proteases, hydrolyses ester bonds in addition to peptide bonds.
During catalysis process of serine proteases, substrate transiently becomes covalently
attached to the enzymes.

Trypsin cuts after Arg, Lys.

Elastase cuts after small uncharged amino acids.

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 11: Membrane Structure and Dynamics
Chapter 12: Membrane Channels & Pumps

Major class of membrane lipids are phospholipids.
Cholesterol is mostly found in plasma membrane than in organelle membranes.

Desensitization: if concentration of acetylcholine remains high, the channel closes
spontaneously.

Depolarization opens the channels.

Conductance as pH .

Gap junction closing is a cooperative effect. (Hill coefficient > 1)

G < 0 passive (can occur spontaneously)
G > 0 active

Mg
++
is required for all ATPases.

CHAPTER 13: SIGNAL TRANSDUCTION CASCADES

E. coli has ~6 flagellin made flagellas. CCW rotation swim. CW rotation tumble.

Rhodopsin --light--> Rhodopsin* Transducin Phosphodiesterase cleaves cGMP into
5GMP

ATP ---- adenylate cyclase----> cAMP + PPi
PPi ----pyrophosphotase----> Pi + Pi

cAMP + H
2
O ----phosphodiesterase---> 5AMP+ H
+

cAMP PKA

PIP2 ----phospholipase C----> IP3 + DAG

PIP
2
= Phosphatidyl inositol-4,5-biphosphate
IP
3
= Inositol-1,4,5-triphosphate
DAG = Diacyl glycerol

IP
3
opens Ca
++
channels
DAG PKC




Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 15: Molecular Motors

Cytoskeleton = microfilaments + intermediate filaments + microtubulues

Synthesis of microtubules occurs by addition of tubulin monomer to the (+) end.
(-) end of the microtubules is bound to the microtubule organizing center.

Kinesin walks from (-) end to the (+) end of microtubules [from center].
Dynein walks from (+) end to the (-) end of microtubules [to center].

ATP is not required for flagellar motion.


Chapter 19: Glycolysis
Control points in glycolysis:

1) Phosphofructokinase (PFK): fructose-6-phosphate ----PFK----> fructose-1,6-biphosphate
(-) ATP, H
+
, citrate
(+) AMP, fructose-2,6-biphosphate

2) Pyruvate kinase (PK): PEP ----PK----> pyruvate
(-) alanine, ATP

Chapter 20: Citric Acid Cycle

TPP is prosthetic group of
- pyruvate dehydrogenase
- -ketoglutarate dehydrogenase
- transketolase
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 23: Protein Turnover and Amino Acid Catabolism

Urea Cycle:

Also known as the ornithine cycle
Takes place only in liver (mammals)
Consists of 5 reactions: 3 cytosolic and 2 mitochondrial
Net reaction: ammonia (NH
3
) is converted to urea
Net input: NH
4
+
, Aspartic acid (Asp), HCO
3
-

Net energy investment: 3ATP
Carrier molecule: ornithine
Arginine: not only an intermediate, but also an activator of the cycle

#1 2ATP + HCO
3
-
+ NH
4
+
carbamoyl phosphate + 2ADP + P
i

#2 carbamoyl phosphate + ornithine citrulline + P
i

#3 citrulline + aspartate + ATP argininosuccinate + AMP + PP
i

#4 argininosuccinate Arg + fumarate
#5 Arg + H
2
O ornithine + urea

Overall reaction:

NH3 + CO2 + Aspartate + 3 ATP + 2 H2O urea + Fumarate + 2 ADP
+ 4 Pi + AMP

The first 2 reactions take place in mitochondria and the last 3 reactions take place in cytosol.

Nitrogen assimilation:
Occurs in plants and algae that cannot do nitrogen fixation.

1. nitrate nitrite ammonia
2. The ammonia is incorporated into glutamine as an amido nitrogen
3. Then is transferred to 2-oxoglutarate to form 2 molecules of glutamate (by glutamate
synthase)
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 31: DNA Structure, Replication and Repair


DNA polymerase I repairs DNA and fills the gaps
DNA polymerase II erases the primers and fills the gaps
DNA polymerase III synthesizes the most DNA

- - - - DNA - gyrase - - adds - negative supercoils- - - - - - -

The following is excerpted from elsewhere.

Fatty acid synthesis.

Acetyl-CoA Carboxylase, which converts acetyl-CoA (input) to malonyl-CoA, is the
committed step of the fatty acid synthesis pathway. This step is also rate-limiting.

AMP-Activated Kinase catalyzes phosphorylation of Acetyl-CoA Carboxylase, causing
inhibition

Summary of fatty acid synthesis (ignoring H
+
and water):
acetyl-CoA + 7 malonyl-CoA + 14 NADPH palmitate + 7 CO
2
+ 14 NADP
+
+ 8 CoA
Summary taking into account ATP-dependent synthesis of malonate:
8 acetyl-CoA + 14 NADPH + 7 ATP palmitate + 14 NADP
+
+ 8 CoA + 7 ADP + 7 P
i


Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 32: Sensory Systems

Response and recovery in smell and vision.

Olfactory sensory neurons

1) Stimulus --> Smell receptor activates a G-protein --> activation of adenylate cyclase
2) cAMP levels rise --> cAMP-gated ion channels open
3) Action potential (nerve impulse)
4) Hydrolysis of GTP of G-protein and its inactivation, Ca++ levels increase
5) Inactivation of adenylate cyclase, Activation of cAMP phosphodiesterase
6) cAMP levels drop
7) cAMP-gated ion channels close
8) Membrane potential repolarizes

* cAMP phosphodiesterase is predominantly found in olfactory sensory neurons.


Vision sensory neurons

1) Stimulus --> Rhodopin activates a G-protein --> activation of cGMP phosphodiesterase
2) cGMP levels drop --> cGMP-gated ion channels (which are open by default when cGMP-
bound) close
3) Action potential (nerve impulse)
4) Hydrolysis of GTP of G-protein and its inactivation, Ca++ levels drop
5) Activation of guanylate cyclase, Inhibition of cGMP phosphodiesterase
6) cGMP levels rise
7) cGMP-gated ion channels open
8) Membrane potential repolarizes


For graphic representations, please click here: Recovery
or copy-paste this URL into your browser"
http://bcs.whfreeman.com/biochem6/pages/bcs-
main.asp?s=00010&n=99000&i=99010.01&v=category&o=|00510|00520|00530|00540|00550|00560|00570|00580|00590|00010|00020
|00030|00040|00050|00060|00070|00080|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000
|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|24000|25000|26000|27000|28000|29000|30000|31000|32000
|33000|34000|35000|99000|&ns=0&uid=0&rau=0
And click to Chapter 32.
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Molecular Biology of The Cell, 4
th
ed., (Alberts, Johnson, Lewis, Raff, Roberts & Walter)












Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 1: Cells and Genomes

Generation of new genes:
- Gene duplication copy of a gene can form and stay inside the same genome as
a related gene
- Mutation DNA changes within genes
- Shuffling segments of genes can be rejoined to form new genes
- Horizontal transfer (intercellular) from one cell/species to another

Vertical transfer transfer of genetic info from parents to progeny

Chapter 2: Cell Chemistry and Biosynthesis

Coenzymes are donors of

ATP Pi
NADH, NADPH H
+
+ e
-

Biotin COOH
Acetyl CoA CH
3
CO
SAM (S-AdenosylMethionine) CH3

A typical covalent bond in a biological molecule has an energy of 15-170 kcal/mole
Average thermal energy at the body temperature is 0.6kcal/mole. Therefore, even an unusually
energetic collision will leave a covalent bond intact.

H-bonds have only about 1/2-1/50 strength of covalent bonds (3-7 kcal/mole).

Clathrate structures (cages) are formed by water molecules around nonpolar hydrophobic
molecules.

In polysaccharides:
16 linkage occurs in branches
14 linkage occurs in all other links

Lipids are defined as water-insoluble molecules that are soluble in organic solvents.
- triglycerides
- phospholipids (major constituents of the cell membranes)
- glycolipids
- steroids (e.g. cholesterol, testosterone)
- polyisoprenoids (long chain polymers of isoprene) (e.g. dolichol phosphate)

Nucleotides:
- DNA, RNA composition
- ATP energy
- CoA donor of acetyl group
- cAMP signaling

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 3: Macromolecules: Structure, Shape and Information

AB A + B
Dissociation rate = dissociation rate constant [AB]

A + B AB
Association rate = association rate constant [A] [B]

At equilibrium:
Dissociation rate = Association rate
dissociation rate constant [AB] = association rate constant [A] [B]
k
off
[AB] = k
on
[A][B]
] ][ [
] [
k
k
K constant eqm
off
on
B A
AB
= = =

Relationship between equilibrium constant and free energy

Standard free energy (G
o
) = -RTln(K)


Chapter 4: How Cells Are Studied

Cell line when a cell type is immortalized and proliferate indefinitely

Cell free system fractionated cell homogenates that maintain biological function

HPLC:
- high-performance (high-pressure) liquid chromatography
- high resolution
- tightly packed tiny particles
- faster: equilibration between the tiny particles takes less time, so solutes can be
separated even at fast flow rates

Fluorescent Analogue Cytochemistry:
e.g. real-time dynamics of rhodamine-tubulin in the cell

Caged molecules: inactive photosensitive precursors of small molecules like GTP.

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 5: Protein Function

N-end rule: relation between the half-life of the protein and its N-terminal amino acid
composition
- Met is stabilizing
- Arginine is primary destabilizing
- arginyl-tRNA-protein transferase (RtPt) links Arginine to the N-term of the proteins bearing
Asp or Glu.



Chapter 6: Basic Genetic Mechanisms

Unusual nucleotides found in tRNA:
1. N,N-dimethyl G
2. Dihydro U
3. N6-isopentenyl A
4. 4-thiouridine

amino acids are ester-linked through their C-term to the 3-OH of the ribose of the tRNAs

aminoacyl-tRNA synthetase proof-reading
peptidyl transferase in 23S rRNA in prokaryotes and in 28S rRNA in eukaryotes rRNA

prokaryotic ribosome:
70S (2.5MDa) = 50S(1.6MDa) + 30S(0.9MDa)
50S = 5S (0.12k) + 23S (2.9k) + 34 proteins
30S = 16S (1.54k) + 21 proteins

eukaryotic ribosome:
80S (4.2MDa) = 60S(2.8MDa) + 40S(1.4MDa)
60S = 5S (0.12k) + 5.8S (0.16k) + 28S (4.7k) + ~49 proteins
40S = 18S (1.9k) + ~33 proteins

Antibiotics acting only on prokaryotes (keep in mind mitochondrial ribosomes):
Tetracycline blocks binding of aminoacyl-tRNA to A-site of ribosome
Streptomycin miscoding and initiation-elongation transition
Chloramphenicol blocks peptidyl transferase (affects mitochondrial ribosomes too)
Erythromycin blocks translocation
Rifamycin blocks RNA polymerase

Antibiotics acting only on eukaryotes:
Cycloheximide blocks translocation (only cytosolic ribosomes)
Anisomycin blocks peptidyl transferase
-amanitin blocks RNA polymerase II

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Antibiotics acting on both:
Puromycin adds to the growing polypeptide chain and causes premature termination
Actinomycin binds to DNA and blocks RNA synthesis

Technique: Chloramphenicol and Cycloheximide can be used to determine whether a protein
is translated in mitochondria or cytosol.

The most frequent DNA damage is deamination and depurination.
Base excision repair:
1) Uracil DNA glycosylase (removes deaminated C (U))
2) AP endonucleases & phosphodiesterase (remove sugar phosphate)
3) DNA polymerase & ligase

Nucleotide excision repair:
1) Nuclease
2) Helicase
3) DNA polymerase & ligase

primosome = DNA helicase + DNA primase

DNA topoisomerase I nicks
DNA topoisomerase II cuts both strands

Nucleosome = repeating structural unit formed by histones bound to DNA.

Genetic recombination:
a) general (by homology, like in crossing-over)
a. RecBCD of E. coli is a helicase and a nicking enzyme (nuclease)
b. RecA has two DNA binding sites: one ssDNA and one dsDNA
c. RecA catalyzes the DNA synapsis
d. RecA is a DNA-dependent ATPase
e. Mismatch proofreading can prevent promiscuous genetic recomb.
b) site-specific
a. conservative: requires some short homology and forms heteroduplex
b. transpositional: no need for homology and no duplex formation
c. enzyme integrase (lambda phage into E. coli genome)

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 7: Control of Gene Expression

Points of gene expression control:
1) Transcriptional control
2) RNA processing control
3) Control of mRNA transport to cytosol and localization
4) mRNA degradation control
5) Translational control
6) Protein activity control

Major groove has more H-bond donor and acceptors and hydrophobic patches on the edge of
the bases. Therefore, DNA-binding proteins recognize the major groove rather than minor
groove.

Insulators:
1) When they flank a gene and its control region, the gene is expressed normally
irregardless of its position in the genome.
2) When they are in between a gene and an enhancer, they prevent enhancer function.

LCR (Locus Control Region): Aside individual control for each of the globin genes, there is a
general control region for the whole family of globins.

Repressors can inhibit by:
1) Competing with activator
2) Masking active site of activator
3) Binding to general transcription factors directly
4) Recruiting chromatin remodeling machinery
5) Recruiting histone deacetylases

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com

Chapter 8: The Cell Nucleus

Essential parts of a chromosome: origin of replication, centromere, telomere

Nucleosome = histone octamer (2H2A, 2H2B, 2H3, 2H4) + 146bp
Nucleosome packing into the 30-nm fiber is mediated by histone H1.

Steroid hormone ecdysone controls gene expression during larval development of Drosophila.

In active regions of chromatin:
1. Histones are present in normal amounts.
2. Histones are highly acetylated.
3. H2B is less phosphorylated.
4. Nucleosomes bind HMG14 and HMG17 two small proteins.

R-G staining of the chromosomes: R: GC-rich, G: AT-rich sequences

Euchromatin replicates early, heterochromatin replicates late.

In nucleolus:
- 45S rRNA precursor is synthesized and processed to 28S + 18S + 5.8S
- 5S is synthesized outside nucleolus, in the nucleus
- Ribosomal proteins are made in cytosol
- Ribosomal subunits are assembled and exported out of the nucleus.

Two large families of transposable elements in primate genomes:
1) L1 transposable element
o Resembles F element (Drosophila) and Cin4 element (maize)
o Encodes a reverse transcriptase
o polyA and 5-truncated
o 4% of human genome

2) Alu sequence
o The most abundant mobile elements in the human genome
o 5% of human genome (500,000 copies)
o 300bp
o Makes target-site duplications when inserts
o Derived from the small cytoplasmic 7SL RNA

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 9: From DNA to Protein

Homeodomain proteins are a special group of helix-turn-helix proteins.

If DNA-binding form of a regulator is turning expression on positive ctrl
If DNA-binding form of a regulator is turning expression off negative ctrl

Trp operon (is repressed when tryptophan is abundant):

- An example of negative control
- Trp repressor is a helix-turn-helix protein
- Trp binds to the repressor and activates it repressor binds to the operator

Lac operon (is activated when lactose is abundant, given that glucose is scarce):
- An example of negative plus positive control
- Allactose binds to the repressor and inactivates it
- Glucose absence leads to CAP binding and thus activation of lac operon

CAP:
- Catabolite Activator Protein.
- cAMP binds and activates CAP.
- cAMP levels rise when glucose levels are low.

DNA flexibility: a curved turn at 200bp
Interaction of two proteins bound on the same DNA molecule is relatively restricted when the
distance between the proteins is 100bp.

Phase variation occurs in Salmonella bacteria. Two different flagellins are flanking one
promoter, which leads to expression of one flagellin or another, depending on its orientation.
This invertible promoter is inherited in the same orientation. The inversions occur rarely and
clones of bacteria will grow up one type of flagellin or another.

DNA methylation ensures that the gene is turned off.
Genomic imprinting requires methylation.
If only paternal gene is transcribed and the maternal gene is silent, the maternal gene is said to
be imprinted.
Mice lacking maintenance methylase die as young embryos.
De novo methylation is done by the establishment methylase.

Sex determination in Drosophila depends on RNA splicing.
The primary signal is the X chromosome / autosome ratio.
Two X chromosomes + two sets of autosomes females
One X chromosome + two sets of autosomes males
This ratio is somehow established early in development and then remembered by each cell
thereafter.

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
mRNA can be localized to specific regions in cytoplasm according to signal in 3UTRs.

Trans RNA splicing:
- occurs in all transcripts in trypanosomes
- all mRNA have a common 5 capped leader sequence

Iron-response element in the 5UTR of ferritin binds aconitase.
Aconitase binding prevents synthesis of ferritin.
Iron binds aconitase and dissociates it from the 5UTR ferritin is made.

Iron-response element in the 3UTR of transferrin receptor binds aconitase.
Aconitase binding prevents degradation of the mRNA and receptors are made.
Iron binds aconitase and dissociates it from the 3UTR transferrin receptor is not made

A common 50 AU-rich sequence in 3UTR (before poly-A tail) promotes the removal of the
poly-A tail and thus rapid degradation.
A repeated sequence in the 3UTR promotes cleavage of the 3UTR by specific endonuclease.

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 10: Membrane Structure

3 Major classes of membrane lipids: phospholipids, cholesterol, glycolipids

Glycocalyx: (cell coat) outer surface of the cell membrane consists of largely carbohydrates.

Spectrin:
Cytoplasmic side of the membrane
220-240kDa and (heterodimer)
Long, thin, flexible rod.

Ankyrin is a peripheral protein that binds to protein band3 and spectrin, therefore attaching
spectrin to the membrane.

Glycophorin: Single-pass transmembrane glycoprotein

Band3 protein: Multi-pass transmembrane anion exchanger (Cl
-
out, HCO
3
-
in) that forms
dimmers.

Porins pass membrane as a barrel.

Ruthenium red or lectins (carbohydrate-binding proteins) can be used to visualize the
glycocalyx.

Selectins: cell-cell adhesion molecules that contain carbohydrate-binding lectin domain

Integrins strengthen cell-cell interaction.
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 11: Membrane Transport of Small Molecules and the Ionic Basis of Membrane
Excitability

Ionophores (permit net movement only downhill of the electrochemical gradient):
1) mobile ion carriers
2) channel formers

Valinomycin: Ring-Shaped Mobile K
+
Carrier
A23187: Divalent Cation Mobile Carrier that takes 2H+ out, per one divalent cation in
Gramicidin A: Channel-Former for Monovalent Cations

Ouabain inhibits Na
+
-K
+
ATPase.

Some Ca
++
-pumps are also membrane-bound ATPases.

Intracellular Ca
++
concentrations ~0.1M
Extracellular Ca
++
concentrations ~1mM

Gibbs-Donnan effect: presence of non-diffusable (across membrane) macromolecules and
metabolites that attract inorganic ions from outside of the cell results in higher concentration of
them inside the cell.

The Gibbs-Donnan effect alters the distribution of diffusable cations and anions across any
membrane where there is a non-diffusable charged species present on one side of the
membrane. If charged nondiffusable species are present on both sides of the membrane then
two Gibbs-Donnan effects are exerted across the membrane. This double Donnan effect is
important in stabilization of cell volume.

What cells do cells do to solve the problem of osmolarity (to prevent deplasmolysis):
1) Animal cells and bacteria pump out Na
+

2) Plant cells have cell wall
3) Many protozoa extrude water from special contractile vacuoles

In cell membranes of animal cells Na
+
is the usual co-transported ion, electrochemical gradient
of which provides the driving force for the active transport of molecules.
In bacteria, yeasts, chloroplasts and mitochondria H
+
rather than Na
+
is usually employed for
this purpose.

Intracellular pH is regulated by two mechanisms: pumping H
+
out, or bringing HCO
3
-
in to
neutralize H
+
.

1) Na
+
-H
+
exchanger (Na
+
in, H
+
out)
2) Na
+
-driven Cl
-
-HCO
3
-
exchanger (Na
+
& HCO
3
-
in, H
+
& Cl
-
out) twice as effective
3) Na
+
-HCO
3
-
symporter (Na
+
in, 2-3 HCO
3
-
out) eletrogenic
4) Na
+
-independent Cl
-
-HCO
3
-
exchanger (Cl
-
in, HCO
3
-
out) prevents too high pH
5) Low pH of lysosomes is maintained by H
+
ATPases
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
In plants, fungi and mitochondria, membrane potential is generated by electrogenic pumps. In
animal cells, however, passive ion movements make the largest contribution.

Resting membrane potential = equilibrium condition, in which there is no net flow of ions
across the plasma membrane.

Nernst equation (not necessary to memorize, just have an idea about it)



V = the equilibrium potential (volts) [= internal-external]
Co and Ci = outside and inside concentrations of the ion
R = gas constant (2 cal mol
-1
K
-1
)
T = absolute temperature (K)
F = Faradays constant (2.3 x 10
4
cal V
-1
mol
-1
)
z = valence (charge) of the ion

Action potential (= nerve impulse) is a traveling wave of electrical excitation.

Myelin is formed by Schwann cells in peripheral nerves and oligodendrocytes in central
nervous system.
Nodes of Ranvier are regularly spaced and almost all Na
+
channels are here.
Saltatory conduction: propagation of action potential along an axon by jumping from node to
node. Advantages: 1) action potentials travel faster; 2) economic (active work is confined to
nodes)

Acetylcholine receptors:
-bungarotoxin binds to acetylcholine receptors and inactivates them.
5 transmembrane polypeptides = 2A+B+C+D = 4 genes
When 2 acetylcholine molecules bind, the channel opens for ~1msec.
Acetylcholine esterase hydrolysises the dissociated acetylcholine.
Na
+
, K
+
& Ca
++
can pass through the channel (not very selective among cations).
Mostly Na
+
ions flow through the channel and cause muscle cells to contract.

Three classes of channel proteins:
4 subunits voltage-gated cation channels
5 subunits transmitter-gated ion channels
6 subunits gap junctions
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 12: Intracellular Compartments and Protein Sorting

Nuclear membrane permeability to proteins:
17kDa 2 min
44kDa 30min
66kDa (globular) hardly at all

Nuclear pore is equivalent to 9nm in diameter, 15nm long cylinder.
Mature cytosolic ribosomes thus (~30nm in diameter) cannot get into the nucleus.
Large proteins like DNA polymerase and RNA polymerase are actively transported into
nucleus.

Nuclear lamina is the inner surface of the inner nuclear membrane.
Nuclear lamina is a meshwork of interconnected protein subunits called nuclear lamins.
Phosphorylation of the nuclear lamins triggers disassembly of the lamina.

Peroxisomes:
- use O
2
to remove H
2
from specific substrates: RH
2
+ O
2
R + H
2
O
2

- contain high levels of catalase and urate oxidase
- catalase uses H
2
O
2
to oxidize substrates: H
2
O
2
+ RH
2
R + 2H
2
O
- -oxidation of fatty acids occurs mainly here (also in mitochondria)
- photorespiration in plant leaves during CO
2
fixation
- glyoxysomes: peroxisome type that converts lipids to sugars in seeds

Glycosylation:
- In ER: N-linked = Asparagine (Asn) linked (to its NH
2
group)
- In Golgi: O-linked = Ser, Thr, hydroxylysine (to their OH group)

Chapter 13: Vesicular Traffic in the Secretory & Endocytic Pathways

GOLGI: Cis face entry
Trans face exit

Cis Golgi has receptors that recognize ER proteins and thus these proteins are brought back to
ER.

Sulfation of proteoglycans gives them their large negative charge.
Sulfate donor is PAPS: 3-phosphoadenosine-5-phosphosulfate

Lysosomal hydrolysases carry a marker mannose-6-phosphate.

LDL (Low-Density Lipoprotein)
1500 cholesterol (in the core) esterified to long-chain fatty acids,
800 phospholipids,
500 unesterified cholesterol molecules, protein

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 14: Energy Conversion: Mitochondria & Chloroplasts

ETS chain:
1) NADH dehydrogenase complex
2) Ubiquinone (e- carrier)
3) b-c1 complex
4) cytochrome c (e- carrier)
5) cytochrome oxidase (aa
3
) complex

Carbon fixation is catalyzed by ribulose biphosphate carboxylase (rubisco).

CO
2
+ ribulose-1,5-biphosphate 2 molecules of 3-phosphoglycerate

3CO
2
+ 9 ATP + 6NADPH + water glyceraldehyde-3-phosphate + 8P
i
+ 9ADP + 6NADP
+



Chapter 15: Cell Signaling

NO (Nitric Oxide):
- is made by NO synthase through deamination of arginine.
- has short half-life (5-10 seconds)
- reacts with iron of guanylyl cyclase (cGMP producing enzyme)

Trimeric GTP-binding proteins (G-proteins): GTP-bound = active, GDP-bound = inactive



Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 17: The Cell Cycle and Programmed Cell Death

Cyclin-Dependent Kinases (Cdks):
Heart of cell cycle control system
Most important regulators of these proteins cyclins
Cdks are active only when bound to cyclins

3 classes of cyclins that are required in all eukaryotic cells:
1) G1/S-cyclins: bind to Cdks at the end of G1 and commit the cell for DNA synthesis
2) S-cyclins: bind to Cdks during S-phase and are required for DNA synthesis
3) M-cyclins: promote the mitosis

In most cells, there is also a G1-cyclin type that is needed to promote the passage though late
G1 phase.

In yeasts, there is only one type of Cdk.
In vertebrates, there are four types.


Cdk activation:
1) Cyclin binding
2) Phosphorylation by CAK (Cyclin-Activating Kinase)













Cdk regulation:
1) Phosphorylation by Wee 1 kinase (inhibition)
2) Dephosphorylation by Cdc25 phosphatase (activation)
3) Cdk inhibitory proteins (CKIs)







Cdk
Cyclin
i i
CAK
Cdk
Cycl
i
P
Cdk
Cycl
i
Inactive Cdk Partially active Fully active
P
Wee1
i
P
Cdk
Cycl
i
Fully active Inactive
P
Cdk
Cycl
i
Cdc25
Phosphatase
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Summary of cell cycle control system:




Major cell cycle regulatory proteins:

A) Kinases and phosphatases
CAK: phosphorylates and activates Cdks
Wee 1: phosphorylates and inactivates Cdks
Cdc25: dephosphorylates and activates Cdks


B) Cdk inhibitory proteins (CKIs)
p27: inhibits G1/S-Cdks and S-Cdks.
p16: inhibits G1-Cdks.
p21: induced by p53; inhibits G1/S-Cdks and S-Cdks.


C) Ubiquitin ligases and their inhibitors

SCF
Targets G1/S-cyclins and certain CKIs that control S-phase entry for proteolysis.
Activity is constant throughout the cell cycle.
Only phosphorylated substrates are ubiquitinated and targeted for proteolysis.

APC (Anaphase-Promoting Complex)
Targets regulators of mitosis for proteolysis (Securin and M-cyclins).
Activated by Cdc20, which is stimulated by M-Cdk.
APC inhibition leads to metaphase arrest.
M-cyclin non-degradability leads to anaphase arrest.
APC activity throughout G1 is maintained by Hct1, which is inhibited by Cdks.
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D) Transcription factors

E2F
Promotes expression of proteins required for S-phase entry.
Inactivated by Rb, which is phosphorylated and inactivated by G1-Cdk.

p53
Promotes expression of proteins inducing cell cycle arrest (especially p21) or apoptosis.
Activated by DNA damage or other cell stress or excessive mitogenic activity.
Targeted for proteolysis by Mdm2.


Chapter 19: Cell Junctions, Cell Adhesion, and the Extracellular Matrix

Cell junctions
Occur in cell-cell and cell-matrix contact in all tissues
Especially plentiful in epithelia

1) Occluding junctions: seal epithelia and prevent even small molecules from leaking
a. Tight junctions (vertebrates only) [zonula occludens]
i. Form a continuous band around each epithelial cell
ii. Require Ca++ for integrity
iii. Major components: Claudins, occludins and ZO proteins

b. Septate junctions (invertebrates mainly)
i. More regular structure than tight junctions


2) Anchoring junctions: mechanically attach cells to other cells or extracellular matrix
General architecture: intracellular anchor proteins + transmembrane adhesion
proteins + (sometimes) intracellular signaling proteins

Adherens junctions & desmosomes
Hold cells together
Transmembrane adhesion proteins CADHERINS

Focal adhesions & hemidesmosomes
Bind cells to extracellular matrix (ECM)
Transmembrane adhesion proteins INTEGRINS

Actin-attachment sites
- Cell-cell junctions (adherens junctions)
- Cell-matrix junctions (focal adhesions)
Intermediate-filament [int. fil.] attachment sites
- Cell-cell junctions (desmosomes)
- Cell-matrix junctions (hemidesmosomes)
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Junctional complex = tight junction + adherens junction (actin) + desmosomes
(intermediate-filaments)

3) Communicating junctions: mediate the passage of signal molecules

a. Gap junctions
Most cells communicate with their neighbors via gap junctions
Small molecules can pass, but macromolecules cant
Connexins and innexins
6 connexins come together to form a connexon
In humans, 14 distinct connexin types - can be homo- and heteromeric.
Can be closed and opened (dynamic structures)
Less regularly organized than junctional complex.

b. Chemical synapses
c. Plasmodesmata (plants only)
Form when smooth ER is trapped across newly forming cell plate
Trapped part of the smotth ER is called desmotubule
Can be made de novo
Can be removed when no longer needed
Molecular weight cut off is similar to that of gap junctions (800Da)
Can be regulated (dynamic).

Cadherins (adherens junctions [actin] and desmosomes [int. fils.]):
- Major molecules responsible for Ca
++
-dependent cell-cell adhesion in vertebrates
- Dimers or oligomers.
- Binding is homophilic.
- Ca
++
-effect is dose-dependent.
- Anchor: Catenin
- Some mediate signals.

Selectins:
- Ca
++
-dependent
- Carbohydrate-binding
- Cell-cell adhesion
- Heterophilic

Extracellular Matrix = GAGs + Fibrous proteins (collagen, elastin, laminin, fibronectin)

Glycose.amino.glycans (GAGs):
- Unbranched.
- Repeating disaccharides (amino sugar + uronic acid repeats).
- Highly negatively charged.
- Forms gels and resist compressive forces.

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Proteoglycans:
- Definition: at least one GAG
- Up to 95% sugar.
- Usually long unbranched GAGs.
- Act as co-receptors.
- Act as signaling molecules.


Chapter 20: Germ Cells and Fertilization

Primordial germ cell: precursors of germ cells that give rise to gametes.

Y-chromosome influences the sex of the embryo by inducing the somatic cells of the genital
ridge to develop into a testis instead of an ovary.

Sry (Sex-determining Region of Y):
expressed only in certain cells in a developing gonad.
induces these cells to differentiate into Sertoli cells.

Sertoli cells produce signal molecules that
promote the development of male characteristics
suppress the development of female characteristics
induce the primordial germ cells to commit to sperm development

Developing egg oocyte.
Mature egg ovum.

Oogenesis:

1) Primordial germ cell (2n)
Enters gonad
2) Oogonium (2n)
Proliferation
3) Primary oocyte (4n)
Arrest in Prophase I
Growth of oocyte
Development of primary oocyte
Formation of egg coat
Formation of cortical granules
Maturation of primary oocyte
Completion of meiotic division I
4) Secondary oocyte + first polar body (2n)
Meiotic division II
5) Mature egg (ovum) + first polar body (n)

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In most vertebrates, oocyte maturation proceeds to metaphase of meiosis II and then arrests
until fertilization. At ovulation, the arrested secondary oocyte is released from the ovary and
undergoes a rapid maturation step that transforms it into an egg that is prepared for
fertilization. If fertilization occurs, the egg is stimulated to complete meiosis.


Nurse cells:
Invertebrates
Some of the progeny of the oogonia become nurse cells instead of becoming oocytes
Usually are connected to the oocyte by cytoplasmic bridges
Macromolecules can pass


Follicle cells:
Accessory cells
Help to nourish developing oocytes
Ordinary somatic cells
Found in both invertebrates and vertebrates.
Arranged as an epithelial layer around the oocyte
Connected only by gap junctions
Exchange of small molecules but not macromolecules.
Secrete macromolecules that contribute to the egg coat, or are taken up by receptor-
mediated endocytosis into the growing oocyte, or act on egg cell-surface receptors to
control the spatial patterning and axial asymmetries of the egg.


Zona pellucida:
A layer of glycoproteins on the surface of unfertilized egg (ovum).
It is usually a barrier to fertilization across species.
Composed of three main glycoproteins: ZP1, ZP2, ZP3.


Sperm DNA is packed with protamines instead of histones.

acrosomal vesicle: Region at the head end of a sperm cell that contains a sac of hydrolytic
enzymes used to digest the protective coating of the egg.

acrosome reaction: Reaction that occurs when a sperm starts to enter an egg, in which the
contents of the acrosomal vesicle are released, helping the sperm to penetrate the zona
pellucida.

The progeny of a single maturing spermatogonium remain connected to one another by
cytoplasmic bridges (syncytium) until they are differentiated into mature sperms.



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Capacitation of sperm:
- sperms must undergo capacitation before fertilization
- capacitation is triggered by bicarbonate ions (HCO
3
-
) in the vagina
- HCO
3
-
ions enter the sperm and activate a soluble adenylyl cyclase enzyme which
produces cAMP
- Capacitation leads to:
o Alteration of lipid and glycoprotein composition of the sperm plasma membrane
o Increase of metabolism and motility
o Decrease of the membrane potential (membrane potential moves to a more
negative potential and the membrane becomes hyperpolarized).

When the sperm fuses with the egg plasma membrane, it causes a local increase in cytosolic Ca
++.

Mechanisms to prevent polyspermy:
1) primary block to polyspermy: rapid depolarization of the egg plasma membrane
2) secondary block to polyspermy: cortical reaction


Cortical reaction:
Started in response to local cytosolic Ca
++
increase after sperm fusion.
Various enzymes stored in the cortical granules are released by the cortical reaction and
change the structure of the zona pellucida, making it harder. As a result, sperm no longer
bind to it.
ZP1 and ZP2 are protelytically cleaved.
ZP3s sugar is detached.


Chapter 21: Cellular Mechanisms of Development

Mid-blastula transition:
- occurs after about 12 cycles of mitosis (~7hrs)
- G1 and G2 phases of cell cycle start to intervene the S and M phases of mitosis (before
mid-blastula transition, there were only S and M phases)
- Transcription of embryo genome begins

Endoderm:
- Digestive system and associated glands
- Liver
- Respiratory system

Mesoderm:
- Connective tissues
- Cartilage, bones, and inner layer of the skin (dermis)
- Muscles
- Cardiovascular system
- Excretory system
- Gonads
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Ectoderm:
- Epidermis, hair, sweat glands, mammary glands
- Nervous system
- Sensory organs

Somites:
o Form during early vertebrate embryo development.
o Paired blocks of mesoderm.
o Lie on either side of the notochord.
o They give rise to the vertebral column, muscles and
associated connective tissue
o Each somite produces the musculature of one
vertebral segment, plus associated connective tissue.
o Myoblasts emigrate from somites.

In mammals, embryonic expression begins at 2-cell stage.
Morula (solid ball of cells) Blastocyst (hollow sphere) Gastrulation

Extraembryonic tissues:
- Amniotic sac & placenta.
- Formed by trophectoderm (wall of the blastocyst sphere).

Neurons are almost always produced in association with glial cells.
Neural tube cells central nervous system (spinal cord, brain, retina)
Cells derived from Neural crest peripheral nervous system


Chapter 22: Histology: The Lives and Deaths of Cells in Tissues

Defining properties of stem cells:
1) Not terminally differentiated
2) Can divide without limit (at least throughout the lifetime of the organism)
3) The daughters of each cell division of stem cells have two choices: differentiate or
remain a stem cell

Scientific problems of dealing with stem cells:
1) They divide very slow hard to study in cell culture
2) Their numbers are very few 1 in 10000
3) They are morphologically very similar to each other hard to find one specific stem
cell we want to study
4) Immune rejection donor-acceptor of the stem cells have to be matched
5) Adult stem cells are not totipotent embryonic stem cells (ES) have to be used, which
is facing ethical problems of dealing with embryos.

Being attached to something is a necessary but not sufficient factor for remaining as a stem
cell.

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Olfactory neurons are constantly (once 1-2 months) renewed by the basal stem cells
sitting just beneath them.
Auditory neurons have to last a lifetime if they are destroyed, they are not replaced.
Retinal neurons also have to last a lifetime, but they renew some of their parts the
rhodopsin stacks.
The lining of small intestine is the fastest renewing tissue.
Hepatocytes divide themselves and they are usually polyploid.
Pericytes are few-numbered connective-tissue family cells that wrap themselves around
small vessels.


VEGF: Vascular Endothelial Growth Factor
HIF-1: Hypoxia-Induced Factor (hypoxia = shortage of oxygen)

Low O
2
High HIF-1 in tissues VEGF expression is induced Tissue cells secrete
VEGF, which acts on nearby endothelial cells to build new blood vessels (angiogenesis)

High O
2
Low HIF-1 in tissues VEGF expression is not induced

Erythropoietin: stimulates production of more erythrocytes in bone marrow.

Myoblasts: precursors of skeletal muscle fibers.
Myoblasts fuse with each other to form myotubes, which are multinucleated long cells which
are then enriched with actomyosin fibers and become muscles.
Muscles grow in length by addition of new myoblasts.
Myostatin: secreted by muscle cells themselves and negatively regulates muscle growth.



Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 23: Cancer

Benign tumor:
- single cluster of cells usually surrounded by a capsule
- cells do not spread
- can be surgically cured

Malignant tumor:
- cells invade other tissues
- new tumors are formed in other tissues that are called metastases

Epithelial tumors carcinomas
Muscle tumors sarcomas
Various hemopoietic cell tumors leukemias

Evidence for clonal origin of cancers:
- Chromosomal aberrations are identical among clones of one cancer
- X-chromosome inactivation evidence that shows that the same X-chr is inactivated in
the clones of one cancer

Replicative senescence: limit on the number of times a certain cell can divide. This number is
usually between 25-50 in fibroblasts, for instance.

Immortalized cell: a cell that divides indefinitely

Key behaviors/skills cancer cells need in general:
1) Disregard proliferative signals (internal and external)
2) Avoid apoptosis
3) Avoid differentiation and replicative senescence
4) Become genetically unstable
5) Escape from home tissues (invasive property)
6) Survive and proliferate in other tissues (gain metastatic potential)

Cancer-causing mutagens:
1) Chemical carcinogens
2) Viruses
3) UV, gamma-rays, alpha-particles (radioactive decay)

Some carcinogens become damaging to DNA only after they have been modified by our liver
enzymes called Cytochrome P-450 oxidases. This is why in the mutagen screen tests,
sometimes (like in Ames test) a liver cell extract is added - to mimic the process of
modification of the chemical in test by liver enzymes.

Tumor initiator:
- mutagenic.
- increases propensity of a cell to become a cancer cell.
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Tumor promoter:
- non-mutagenic.
- causes neoplastic growth in cells that were previously exposed to tumor initator.
Cells of a tissue initiator+promoter Papillomas

Viruses can be both initiators and promoters.

Proto-oncogene dominant mutation (Gain of Function) Oncogene
Tumor suppressor gene (TSG) recessive mutation (Loss of Function) Inactive TSG

Oncogenes can be identified when they are added to cells (a cancer phenotype would be
expected). TSGs can be found when both copies are removed.

Loss of heterozygosity: deletion of one chromosomal region that can be detected by
polymorphic markers.

3 ways to convert a Proto-oncogene to Oncogene:
1) Point mutation or deletion
2) Gene amplification
3) Chromosomal rearrangement that leads to a) overexpression; b) fusion of proteins

6 ways to lose a TSG:
1) Nondisjunction (chromosome loss)
2) Nondisjunction (deletion)
3) Mitotic recombination
4) Gene conversion
5) Deletion
6) Point mutation







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Genetics, 3
rd
ed., Weaver and Hedrick










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Chapter 6: Chemistry of The Gene

Genes are extremely large informational molecules.

Property A-DNA B-DNA Z-DNA
Humidity 75% 92%
Base inclination 20
o
0
o
7
o

Residues / turn 11 10 12
Pitch (nm) 2.8 3.4 4.5
Right handed Right handed Left handed
dsRNA, DNA-
RNA hybrids,
dehydrated DNA
most commonly
found DNA (in cells)
Poly pur-pyr

T
m
: temperature at which DNA strands are half-denatured

The more complex is DNA, the higher is its C
o
t value.

10bp = 34A
o

1bp = 660g/mol

Electrophoretic mobility of larger DNA strongly deviates from linear dependence to the DNA
length.

DNA size can be measured by 1) Electrophoresis
2) Electron microscopy
3) Ultracentrifugation


C-value = DNA content / haploid cell.



Chapter 7: Replication and Recombination of Genes

The complementarity governs the replication of DNA.

DNA polymerase I :
1) 5'-3' polymerase activity
2) 3'-5' exonuclease activity (proofreading mechanism)
3) 5'-3' exonuclease activity (removes primers)

DNA replication is semiconservative, semidiscontinuous and usually bidirectional.

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Replicon: DNA under control of one origin of replication. Some viral and bacterial genomes
have only one origin of replication => replicon genome.

Enzymes that function in DNA replication:
1) Helicase: uses 2 ATP molecules to open up 1 bp.
2) SSB: prevents reassociation (binds cooperatively; stronger to ssDNA than to dsDNA)
3) DNA gyrase (topoisomerase II): releases strain by breaking both DNA strands and
resealing them. Pumps negative supercoils to counteract with positive ones, which are
introduced during DNA unwinding by helicase.
4) Primosome: large aggregate (about 20 polypeptides) that makes the primers in E. coli
(=helicase+primase).
5) Primase: primer synthase
6) DNA polymerase III:
a. 10 different polypeptides.
b. Proofreading activity
c. Processive

Processivity: tendency to stick to the job once it starts.

Mammalian DNA polymerase:
1) : synthesis of lagging strands
2) : DNA repair
3) : mitochondrial DNA synthesis
4) : synthesis of leading strands
5) : function unknown, similar to

Phage Lambda: rolling circle replication = sigma replication.

Telomerase:
1) Adds DNA to 3'-end of a chromosome
2) Contains an RNA molecule in its structure

Usually primers are made of RNA: DNA replication of phage M13 inhibited by rifampicin.

Why primers are made of RNA?
Primers are made without proofreading. Hence, they accumulate more errors (mutations).
Being made of RNA ensures that they are recognized and degraded and replaced.

RNA genomes can't grow too large. Because
1) No proofreading mechanism => many mutations are accumulated
2) Extra -OH group makes RNA less stable, easily degradable.

Consequences of proofreading
1) NO DNA polymerase that can polymerize in 3'-5' direction
2) Primers are needed to initiate polymerization.

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From the intrinsic properties of dNTPs:
If polymerization is 5'-3' => the energy source is the incoming dNTP.
If polymerization is 3'-5' => the energy source is the nucleotide at the 5'-end.



Chapter 8: Transcription and Its Control in Prokaryotes.

Transcription:
1) Initiation
2) Elongation
3) Termination

Transcription is asymmetric.

Core only (no ) RNA polymerase holoenzyme
Non specific Specific
Symmetric Asymmetric

Operon: group of contiguous, coordinately controlled genes

Lac operon:
LacI: encodes a repressor monomer
P: promoter: RNA polymerase binds here
O: operator: tetramer repressor binds here
LacZ: beta-galactosidase
LacY: glucoside permease
LacA: glucoside transacetylase
Allolactose: inducer
Glucose: catabolyte repression
Glucose/cAMP/CAP: positive control (Glucose dec. => cAMP inc. => cAMP binds CAP =>
cAMP*CAP bind promoter and facilitate RNA polymerase binding.)

trp operon:
trp R: aporepressor monomer
O,P: operon/promoter: dimer repressor binds here
Leader/Attenuator: premature termination of transcription if trp is abundant.
TrpEDCBA: trp synthesis pathway enzymes
Trp: corepressor

[Trp] inc. => trp binds to aporepressor => repressor dimerizes => binds to operator => RNA
polymerase cannot pass and transcribe the trp operon genes.

Termination of Transcription:
1) terminating hairpin loop
2) rho factor + hairpin loop
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Temporal Control of Transcription:

Temporal Control of Transcription in SPOI-infected B. subtilis:

Early transcription:
host sigma + RNA polymerase core => early proteins including gp28
Middle transcription:
gp28 + RNA polymerase => middle proteins including gp33, gp34.
Late transcription:
gp33, gp34 + RNA polymerase => late proteins

Temporal Control of Transcription in T7-infected E. coli:
1) host sigma + RNA polymerase core => class I proteins including new RNA polymerase
2) new RNA polymerase => class II ans class III proteins

Temporal Control of Transcription during lytic infection by phage:

phage genome

att int xis cIII t1 N O
L
P
L
cI P
RM
O
R123
P
R
P
RE
cro t1 cII O P Q t2 P'
R
S t3 R

Immediate early transcription: green
Delayed early transcription: magenta
Late transcription: not shown for simplicity

CI: repressor (provides lysogeny)
CII: stimulates polymerase to bind to P
RE
(for lytic cycle)
CIII: slows down degradation of CII by proteases
N: antiterminator (anti-t1)= permits delayed early transcription
Cro: antirepressor: binds to all rightward and leftward operators (for lytic cycle)
P,O: necessary for phage replication
Q: antiterminator (anti-t2) = permits late transcription
P
RE:
repressor establishment promoter
P
RM:
repressor maintenance promoter
t: terminator
P
'R:
late transcription promoter


CI binds to O
R
and

O
L
=>

=>

cro is turned OFF since RNA polymerase can't bind P
R
=>

N is turned OFF since RNA polymerase can't pass after O
L

=> NO gene except CI is transcribed
=> lysogenic phase starts

LYSOGEN: bacterium that is in lysogenic phase
PROPHAGE: phage DNA in lysogenic phase

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A LYSOGEN IS IMMUNE TO SUPERINFECTION BY ANOTHER PHAGE THAT HAS
THE SAME CONTROL REGION THE PROPHAGE HAS.

The battle between cI and cro:
If cI wins => lysogenic phase begins
If cro wins => lytic phase begins

Viruses and bacteria in a plaque are genetically identical => decision lytic/lysogenic decision is
not genetic.

Starvation => low level of proteases => high level of CII => lysogeny
Rich medium => high level of proteases => low level of CII => lytic

Lysogeny is cheap = it only requires synthesis of small repressor

cro binds to O
R3
=> RNA polymerase cannot bind P
R
=> cI not transcribed => NO lysogeny

cro binds to all rightward and leftward operators => NO transcription of delayed early genes
=> NO cII and cIII => NO lysogeny

Lytic phase can be induced by radiation or mutagens.

Radiation => SOS response of E. Coli => RecA => co-proteolytic activity => repressor
cleaves itself => RNA polymerase can bind P
R
and pass O
L
=> lytic phase starts



Chapter 9: Eukaryotic Gene Structure and Expression

Nucleosomes = histones + 200bp DNA => 1
st
order of chromatin folding (coiling, condensing)

Solenoid (30-nm) = 2
nd
order of chromatin folding (H1 is playing role in formation)

Brush-like structure = 3
rd
order of chromatin folding

Heterochromatin: condensed, transcriptionally inactive
Euchromatin: extended, at least potentially active

RNA polymerase I: rRNA precursors (large rRNAs)
RNA polymerase II: mRNA precursor
RNA polymerase III: tRNA precursor, 5S rRNA precursor

Transcription factors:
1) General (class II factors)
2) Gene-specific (gene activators)

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Classical RNA polymerase III genes: ICR (Internal Control Region) {5S rRNA, tRNA}
Non-Classical RNA polymerase III genes: 5'-flanked promoters
{7SL RNA}

Enhancers:
1) Stimulate transcription from promoters
2) Orientation and position independent
3) 72-bp-repeats in SV40 virus
4) Usually tissue specific depend on transcription factors
5) No real consensus sequence

Silencers:
1) Inhibit transcription
2) Depend on silencer proteins
3) Also work at distance as do enhancers


TFIID = TATA box Binding Protein (TBP) + TBP-Associated Factors (TAFs)

Class II pre-initiation complex:
Promoter (NO TATA box = core) +D+B+[F+RNA polymerase II]+E+H+A

Class I pre-initiation complex:
Promoter + SL1 + UBF + RNA polymerase
UBF: Upstream Binding Factor (binds to UCE => provokes RNA polymerase to bind)
UCE: Upstream Control Element
SL1: strengthens the RNA polymerase's binding (TBP-containing factor)

Class III pre-initiation complex:
Promoter (NO TATA box) + TFIIIC {only=tRNA}+ TFIIIB {+TFIIIA = 5S rRNA}+ RNA
polymerase III

Structure of gene-specific transcription factors:
1) DNA binding domain:
a. Zinc fingers: -helix = DNA recognition element
b. Homeodomains
c. BZIP domains: DNA-binding and dimerization domains are inseparable.
2) Transcription activating domains
a. Acidic
b. Glutamine-rich
c. Proline-rich

Gene-specific factors bind to
1) Promoter elements (GC boxes, CCAAT boxes)
2) Enhancers

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Transcription factors bind to DNA as dimers or tetramers because this allows cooperative
binding, which increases the affinity between protein and DNA, and permits higher sensitivity.


The Relationship Between Gene Structure And Expression

Sharp and Roberts, 1977 made R-looping experiments: hybridize RNA to its DNA template

RNA-DNA hybrids are more stable than DNA duplexes.

Why do we use viruses in genetic research?
Viruses are much simpler genetically than their hosts much easier to study.
DNA viruses like adenovirus and SV40 use the same gene expression machinery as
their host.

RNA Splicing

Alternative Splicing
Self-Splicing

hnRNA = heterogeneous nuclear RNA = precursor of mRNA
large;
only in nucleus;
turns over rapidly

Cryptic sites: splicing signals used only when the normal sites are mutated.

Spliceosome: Complex and large particle (50-60S) on which the splicing occurs
mRNA precursor + set of snRNPs (U1-U6)

Splicing Schemes:
Group I: used G to initiate self-splicing
Group II: does not need G; uses A within the intron itself to initiate self-splicing
Nuclear mRNA: uses A within the intron itself + spliceosome

RNA processing:
Trimming RNA precursor outside the exons
Methylating
Adding nucleotides to 3 end
Blocking the 5 end with specific structure called cap

Darnell et al. used ribonuclease A (cuts after C and U) and ribonuclease T1 (cuts after G) to
release intact poly(A)



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Poly(A):
3 end
150-200 nt
poly(A) polymerase
posttranscriptionally or cotranscriptionally
found on hnRNAs also
function1: protect from cytoplasmic RNases
function2: stimulate translation

Once mRNA enters cytoplasm, its poly(A) tails is constantly being degraded by RNases and
rebuilt by cytoplasmic poly(A) polymerase.

Addition of poly(A) tail to the 3 end of an RNA can occur sometimes while transcription is
still in progress:

[1] Clipping enzyme clips mRNA upstream after signal for this enzyme: AAUAAA,
[2] Poly(A) polymerase starts adding poly(A).

Cap:
[1] Phosphotransferase removes the 5 terminal P leaving diphosphate group
[2] Capping enzyme adds GTP to 5 end with a 5-5 triphosphate linkage
[3] Methyl transferases transfer Me groups from SAM (S-adenosylmethionine) to
appropriate positions on the cap.

Capping, also, occurs before transcription is complete. (cotranscriptional process)

Function1: protect mRNA from degradation by RNases from 5 end
Function2: necessary for binding mRNA to ribosome



Chapter 10: Translation

Ribosome = rRNA + Proteins

Ribosome is a self-assembly system, it can be reconstituted from its own component parts
without any enzyme or factors.

E. coli ribosome (70S):
50S = 23S and 5S rRNA + 34 proteins
30S = 16S rRNA + 21 proteins
50S + 30S = 70S

Polysome (polyribosome): many ribosomes sit on one mRNA molecule.


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t-RNA:
the adapter molecule
aminoacyl-tRNA synthetase (alanine-tRNA synthetase)
cloverleaf model - secondary structure
L-shaped 3D structure
contains many modified bases

structure:
anticodon, anticodon loop, variable loop
TC loop ( = pseudouridine)
acceptor stem (terminal CAA)
D-loop (D = dihydrouracil loop)

Aminoacyl-tRNA synthetase:

amino acid + ATP aminoacyl-AMP + P
i
P
i
(activated amino acid)
aminoacyl-AMP + tRNA aminoacyl-tRNA

Each organism has only 20 aminoacyl-tRNA synthetases aminoacyl-tRNA synthetases are
very specific
Recognition elements: acceptor stem (discriminator base: NCCA)

genetic code: the set of codons in mRNA that stand for the 20 amino acids in proteins.

One base substitution (missense mutation) leads change in only one amino acid
=> the code is nonoverlapping (each base is part of only one codon).

Shift of the reading frame by deletion or insertion of a single base (frameshift mutations)
=> no gaps in the code (each base is a part of codon, no commas).

Frameshift effect by single base deletion is neutralized by single base insertion and vice versa.
Insertion or deletion of three bases (also 6, 9, 12, etc) does not lead to frameshift effect.

The code is degenerate: more than one codon codes for a given amino acid.

Wobble hypothesis (Francis Crick):
The third base of a codon can form unusual base pairs. (violating the Watson-Crick
base pairing rules).
Inosine (I) can base pair with C, U, or A.
Wobble pairs are G-U and I-A.
The number of tRNAs needed to translate the genetic code is reduced. (both UUU and
UUC code for phenilalanine but one type of tRNA (3AAG5) is enough to translate
both codons).

The genetic code is not strictly universal. There are some deviant codon examples (e.g.
mitochondrial genetic code is slightly different)
Both reactions
catalyzed by
aminoacyl-tRNA
synthetase
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MECHANISM OF TRANSLATION:

INITIATION:
In prokaryotes the first amino acid is always N-formyl methionine (fMet).
There is a special tRNA called fMet-tRNA. (tRNA
f
Met
)
First normal Met binds to fMet-tRNA and then Met is converted to fMet
In eukaryotes normal Met and special tRNA
i
Met
is used for initiation
Met is removed from the proteins usually if not always.
fMets formyl group is always removed.
Codon for initiation in both prokaryotes and eukaryotes is AUG (or sometimes GUG).
In prokaryotes Shine-Dalgarno sequence (5-AGGAGGU-3) just at the 5 side of the
initiation codon is more or less complementary to the 3 end of 16S rRNA in the
ribosome => this sequence helps the binding of ribosome to the initiation regions of
mRNA.
In eukaryotes 5-cap ensures binding of mRNA to ribosome.
Leader: the RNA between the cap and the initiation codon.

Proteins grow in N to C direction.
5-end of an mRNA encodes the amino end of a protein.

ELONGATION:
1) Elongation factor needed for an aminoacyl-tRNA to bind to aminoacyl (A) site: EF-Tu +
GTP (eEF-1)
2) Peptidyl transferase (inhibited by chloramphenicol)
3) Elongation factor needed for translocation from A (aminoacyl) to P (peptidyl) site: EF-G +
GTP (eEF-2)

TERMINATION:
In Prokaryotes: translation release factors: RF-1, RF-2, RF-3 + GTP
In Eukaryotes: eRF + GTP

Translational control:
Secondary structure of mRNA
Feedback of the ribosomal proteins

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com

Chapter 11: Gene Mutation

Classification of gene mutations according to their effect on phenotype:
1) Germ-line or germinal mutations
2) Somatic mutations
3) Morphological or visible mutations
4) Nutritional mutations (prototrophs-auxotrophs)
5) Lethal mutations
6) Conditional mutations

Classification of gene mutations according to their effect on genetic material:
1) Missense
2) Nonsense
3) Framehift

Transitional mutations: Pyr -> Pyr or Pur -> Pur
Transversional mutations: Pur -> Pyr or Pyr ->Pur

Transversional mutations are more drastic because:
1) Chemically: purine resembles purine and pyrimidine resembles pyrimidine
2) Genetically: the degeneracy of the genetic code: related codons ending with the same
base type are more likely to code for the same amino acid.

Spontaneous Mutations
1) Gene replication machinery fault
2) Tautomerization (amino-imino, keto-inol)
3) Frameshift by looping out
4) Deamination (C->U, A->hypoxanthine, 5-methylcytosine->T)
5) Triplet repeats

Mutation rate = number of mutations / generation / cell division / number of gametes

Chemical mutagenesis
1) Nucleoside analogues (5-bromodeoxyuridine: BrdU): resembles T, can occasionally
switch to enol tautomer => BrdU
.
A -> BrdU
.
G
2) Alkylation of bases: by ethyl methane sulfonate (EMS) as many carcinogens
3) Deamination: by nitrous acid, bisulfite
4) Intercalating agents: acridine dyes, ethidium bromide
Radiation-induced mutagenesis
1) UV: relatively weak, causes Pyr-Pyr dimers, strongest at 260nm.
2) -rays and X-rays: strong, ionizing radiation, by ionizing molecules around DNA
(especially water molecules) to form free radicals, can break chromosomes = clastogen.
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
DNA repair
a) Directly undoing the DNA damage

Photoreactiation by photoreactivating enzyme: DNA photolyase
T
V
T ---> T
.
T

O
6
-methylguanine methyl transferase: suicide enzyme
G-Me ---> G

b) Excision Repair
Base excision repair:
DNA glycosylase -> Ap endonuclease -> DNA polymerase I -> DNA lygase

Nucleotide excision repair:
Excinuclease (uvrABC endonuclease) -> DNA polymerase I -> DNA lygase

c) Mismatch repair
Question: Which strand is paternal and which strand is to be repaired?

In E. coli:
Parental strand has identification tags (A-Me) every 250bp
Methyl transferase methylates A of GATC
Mismatch repair system takes advantage of the time when newly synthesized strand is not
methylated yet and repairs it according to the parental strand, which is methylated.

d) Coping with DNA damage without repairing it
Recombination repair: requires DNA replication
Error-prone repair (part of SOS response):
RecA --
UV
--> RecA co-protease
LexA -> cleaved LexA
Inactive umuDC operon -> active umuDC operon
Stalled replication -> Replication goes on

Wild-type E.coli are tolerant to ~50 Pyr-Pyr dimers.

Ames test:
Special bacterial strains of Salmonella are plated on Petri dishes with LB-agar that contains
potential mutagenic chemical. If the chemical is really mutagenic, the bacteria will undergo
mutations and there will be revertants colonies of bacteria that mutated and became
resistant to certain restrain put in the LB-agar.

Modifications:
1) liver extract - to mimic metabolism
2) leaky cell walls - to allow mutagens to enter the bacteria
3) plasmid with aberrant umuDC - extra efficient error-prone SOS repair
Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 12: Transposable Elements

BACTERIAL TRANSPOSONS
1) IS1: simplest
2) Tn3: at least one more gene beyond the ones needed for transposition
3) Tn9: complex
4) Mu: can transpose in its prophage phase

Insertion Sequences (IS):
The simplest transposons (Shapiro, 1960s)
Short inverted repeats at ends (Cohen, stem-loop structure under microscope,1960s)
Contain only elements necessary for their own transposition
At least two genes coding for an enzyme: transposase
Transposition causes duplication of short sequence in the target DNA, so that after
transposition there is a copy of the short sequence flanking the IS.
Sometimes called selfish DNA
Sometimes called jumping DNA

Transposition
1) Conservative (cut-paste)
2) Replicative (copy-paste)

Illegimate recombination: little dependence on the DNA sequence transposed

Tn3: IR---tnpA---tnpR---res---bla---IR
tnpA: transposase
tnpR: resolvase
bla: -lactamase = ampicillin resistance gene (Amp
r
)
IR: Inverted Repeats
Res: resolution site





Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Evidence for Tn3 two-step transposition:
1) tnpR mutants cannot resolve cointegrates
2) tnpR provided by a host chromosome or a plasmid helps to resolve the cointegrate

Bacteriophage Mu can behave as a transposable element.

EUKARYOTIC TRANSPOSONS
1) Ac/Ds: activator/dissociation in maize
2) Ty: in yeast
3) Copia: in Drosophila
4) Retroviruses:

1940s McClintock => Fedoroff: Ac/Ds

1970s: Teminn and Baltimore: reverse transcriptase
retrotransposone = retroposon


Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Chapter 13: Genetics of Bacteria and Phages

Escherichia coli Homo sapiens
4.2x10
6
bp 3x10
9
bp
3000 genes 100.000 genes

Selection vs Screening:
Selection for trp
+
strain
Screening for trp
-
strain (by replica plating)

1946 Lederberg and Tatum:
Conjugation = direct contact between bacterial cells via sex pilus (F pilus).

Hayes et al.: one-way DNA transfer during conjugation:
1) Streptomycin treated donors (F+) could still participate in conjugation, but
streptomycin treated recipients (F-) could not.
2) If F+ and F- differed in several loci, the recombinant had mostly the recipients genes.

F-factor:
A 94,5kb plasmid: F-plasmid
F- F+ at high frequency

A new type of E.coli in addition to F- and F+: Hfr
Hfr:
High frequency recombinants
Mutant F+ strains
Occasionally, F+ recombines with its host's chromosome => F+ --> Hfr
Cannot transfer F-plasmid at high frequency
Instead, transfer host chromosome genes at high frequency

1950's: Woolman & Jacob used fluctuation test devised by Delbrck & Luria to test whether
the F+ Hfr transition is equilibrium or is permanent.
1. Take very dilute F+ culture (250cells/ml)
2. Separate in two parts
3. Separate one of the parts into 50 subcultures
4. Test for the ability to for recombinants
Non-mutational hypothesis: If F+ <--> Hfr is correct => each of the 50 subcultures should
show equal proportion of Hfr
Mutational hypothesis: If F+ -mutation-> Hfr is correct => each of the 50 subcultures
should vary widely in the proportions of Hfr

F' = F-plasmid + some host DNA (F-lac plasmid = F-plasmid + lac operon)

The introduced DNA from donor to recipient (e.g. lac operon) should be integrated by
recombination.

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
Transduction: use of phages to carry bacterial genes into new host cells.

Specialized transduction: phage DNA carries bacterial genes
Generalized transduction: phage heads carry bacterial genes instead of phage genes
Temperate phages: do not kill the hosts (lytic infection), e.g. phage
Virulent phages: kill their hosts (lysogenic infection), e.g. T2, T4 phages

During lysogenic infection phage usually inserts its genome at a site called attB between
gal and bio genes in E. coli. Usually the phage DNA excises perfectly but sometimes it
comes out with a piece of host DNA.

Copyright 2005-2007 Ayaz Najafov www.GREBIO.com
REFERENCES


1. Biochemistry by Berg J.M., Tymoczko J.L., Stryer, L. (2002, 5
th
edition)
2. Molecular Biology of the Cell by Alberts B. et al. (2002, 4th edition)
3. Genetics by Weaver R.F. and Hedrick P.W. (1997)
4. Biochemistry by Voet D and Voet J (1995, 3
rd
edition)
5. http://en.wikipedia.org/wiki/Fischer_projection