Information flow involving DNA, RNA,

and proteins within cells

“CENTRAL DOGMA OF MOLECULAR

GENETICS”
 MUST BE KNOWN
Genome size, estimated gene number, av.
gene density and chromosome number in

E. coli, yeast, Arabidopsis thaliana, maize,

Caenorhabditis elegans, Drosophia
melanogaster, zebra fish and human
YOUR COCLUSION WHEN COMPARED?
 Concept of Inheritance:Brief
History
• Nucleic acids or proteins? (Late 19th century)
• Proteins favored (until the 1940s)
• P. A. Levene (1910): Tetranucleotide hypothesis
• E. Chargaff (1940): Levene’s 1:1:1:1 ratio was incorrect!
• F. Griffith (1927): Transformation experiment
• O. Avery, C. MacLeod, M. McCarty (1944): direct
experimental proof that DNA, but not protein, is responsible for
heredity
• A. Hershey and M. Chase (1952): convincing evidence
• Rosalind Franklin (1950-1953): X-ray diffraction analysis;
some sort of helix, but no definite model proposed
• James Watson and Francis Crick (1953): published double-
helical model (Nature)
 Griffith’s transformation experiment
(1927) with Diplococcus pneumonia

“Transformation
principle”

with capsules (type IIIS), without capsules (type IIR)

Summary of Avery, MacLeod, and McCarty’s
experiment (1944)

EtOH ppt→N/P ratio→coincided with that of Na desoxyribonucleate

Life cycle of a T-even bacteriophage
Summary of the Hershey–Chase
experiment (1952)

Convincing evidence!
X-ray diffraction analysis
Precursor during NA biosynthesis
+cell bioenegetics
(a) Linkage of two nucleotides by the
formation of a 5' phosphodiester bond,
producing a dinucleotide. (b) Shorthand
notation for a polynucleotide chain
A weak electrostatic interaction between
a covelently bonded H atom and an atom wth an
ushared electron pair
 TWO RULES

*A+G=C+T

*A+T/G+C ratio varies

greatly among species
 The DNA double helix as proposed by
Watson and Crick (B-form)

A detailed
view
1 Angstrom
(Ā)=0.1 nm)

Each complete turn: 360o

Antiparallel
nature
Mirror images of
one another
B-DNA is the most common at neutral pH and physiological salt concentrations
 Different Conformational Forms
• A-DNA: high salt and dehydration cond., a thicker right
handed duplex. More compact; 11 bp each turn, 23Ao in
diameter, major and minor grooves appear different
• C-DNA: greater dehydration cond., 9.3 bp per turn, 19Ao in
diameter, right-handed
• D-DNA and E-DNA: in helices lacking G, 8 and 7 bp each
turn, right-handed
• Z-DNA: its bases seem to zigzag; left-handed. One turn 4.6
nm, 12 bp; DNA with alternating G-C only; no major
groove
• P-DNA: More narrow; phosphate groups inside; 2.6 bp per
turn
Different forms: Molecular recognition for proteins
during replication and transcription?? Awaits
demonstration in vivo.
 The 4 types of RNA (size, function,
sedimentation behaviour*)

• rRNA: at least 80 %; 5S (P, E), 5.8S (E), 16S

(P), 18S (E), 23S (P), 28S (E)
• mRNA: 5 %; varies (P and E)
• tRNA: 15 %; 4S (P and E)
• snRNA (small nuclear): mRNA processing (RNA
splicing); referred to as small nuclear
ribonucleoproteins (SNRNP)
• Telomerase RNA: maintenance of the telomeres or
chromosome ends
• Antisense RNA, miRNA, siRNA: gene regulation
*density, mass, shape; its measure is Svedberg
coefficient (S)
Analytical techniques to
investigate DNA and
RNA
 UV absorption

At 254-260 nm most strongly, due to

interaction btw UV light and ring systems of
bases.

To localize, isolate and characterize.

 Sedimentation behavior: Separation of a
mixture of two types of nucleic acid by
gradient centrifugation

Sedimentation Equilibrium
Centrifugation (Density
Gradient Centrifugation):
Neutral buoyant density;
no further migration

To fractionate the gradient, successive samples are eluted from the bottom of
the tube. Each is measured for absorbance of UV, producing a profile of the
sample in graphic form.
(GC bp.s are more compact and dense)
Increase in UV absorption vs temperature
(the hyperchromic shift) for two DNA
molecules with different GC contents (melting
profile)

Lesser GC
content
Tm: Melting
temp.

Greater GC
content

Viscosity decreases, but UV absorption, buoyant

density increases
 DNA reassociation time course=
Reassociation kinetics

k: second-order
rate constant

*Co: initial conc. of ssDNA

*Note that the abscissa (C0t) is plotted logarithmically
Cot Analysis I
 Cot Analysis II

Cot1/2: Half reaction time; when one half of the DNA is present as ds fragments
(directly proportional to genome size; useful to assess genome size of organisms)
Cot Analysis III
Molecular hybridization between
DNA fragments and RNA

Probes, Southern blot

Hybridization,
Autoradiography
 Detection

• The probe DNA is labelled so that it can be detected,

usually by incorporating radioactivity or tagging the
molecule with a fluorescent or chromogenic dye.

• After hybridization, excess probe is washed from the

membrane and the pattern of hybridization is
visualized on X-ray film by autoradiography in the
case of a radioactive or fluorescent probe, or by
development of color on the membrane if a
chromogenic detection method is used.
 Fluorescence in situ Hybridization
(FISH)
• used to identify the presence and location
of a region of DNA or RNA within
chromosome preparations, fixed cells or
tissue sections
• means you can view a segment or entire
chromosome with your own eyes
• M phase chromosomes oftenly
 FISH Procedure

↓
Localize to chromosomes with microscopy. Or after hyb. with biotinylated
probe, fluorescein coupled to avidin is reacted with cytological specimen
FISH
Electrophoretic separation
Semiconservative replication of DNA
The Meselson–Stahl experiment (1958)

(NH4Cl as the only N source)

The results were as expected..
 Bidirectional replication of the E. coli
chromosome
Ca. 300 bases
recognized by sp. initiation
proteins

(arrows identify the advancing replication forks moving in

opposite directions)
 Processivity

• Amount of polymerization catalyzed by an

enzyme each time it binds to a template.
• Measured in nucleotides polymerized per
initiation
• High processivity of DNA Pol III results
from activities of non-polymerase subunits
 DNA Polymerase III
The holoenzyme (active enzyme): An asymetrical dimeric
complex arranged from combination of 10 distinct subunits.
Three subunits form the core of the enzyme, these are:
alpha (α): largest subunit; 5’→3’ polymerization,
epsilon (ε): 3’ →5’ proofreading, and
theta (θ): stimulates ε-exonuclease
gamma (γ) complex: delta, delta prime, chi, gamma, psi, and
tau; variationally binding to this core, and conferring the
full functions and characteristics the enzyme needs to carry
out the replication of DNA; loading the enzyme onto the
template requiring hydrolysis of ATP
beta (β): sliding clamp
 Subunits of DNA Pol III and functions
Functional Mass
component Subunit (kDa) Gene Activity
Core polymerase a 130 polC (dnaE) 5' to 3' polymerase
e 27.5 dnaQ (mutD) 3'-5' exonuclease
q 10 holE Stimulates e exonuclease
Gamma complex t 71 dnaX Dimerizes cores
(Clamp loader/ g 45.5 dnaX Binds ATP
ATPase) d 35 holA Binds to b
d' 33 holB Binds to g and b
c 15 holC Binds to SSB
y 12 holD Binds to c and g
Sliding clamp b 40.6 dnaN Processivity factor
 5' to 3' synthesis of DNA

Always proceeds from the end with 5’-P to the 3’-OH end (5’-P of incoming nucleotide is
attached to 3’-OH of the previously added nucleotide)
Helical unwinding of DNA during replication as
accomplished by DnaA, DnaB, and DnaC
proteins (helicases)

*Double helix is unwound; small ss region is formed.

*As unwinding proceeds, DNA topoisomerase II (DNA gyrase) relaxes
DNA.
The initiation of DNA synthesis

(10-12 nts long;

providing free 3’-OH
groups; synthesized
by primase)
 Opposite polarity of DNA synthesis
along the two strands

*Okazaki fragments: ca. 1000 bases

long
*Leading strand is primed only once; at
the origin
 How concurrent DNA synthesis is achieved on
both the leading and lagging strands at a
single replication fork?

The lagging template strand is looped in order to invert the physical (not biochemical!)
direction of synthesis
Summary of DNA synthesis at a
single replication fork

(stabilize ss
DNA)

(11 bases)

Proofreading: 3’→5’ exonuclease activity of DNA

pol III.
100 X increase in fidelity of synthesis: Error rates
during DNA synthesis are in the 10–6 to 10–8 range
Sealing two fragments on the lagging
strand

5’→3’ exonuclease activity of

DNA polymerase I
 Assembles at the
replication fork:

Primosome: protein complex that

initiates synthesis of a DNA
strand.

Replisome: complex of proteins

engaged in elongation of the
newly synthesized DNA strand.
As big as ribosome at the
replication fork.
SUMMARY of steps in E. coli DNA synthesis:

•DnaA, DnaB, DNAC (helicases) and ATP bind to replication

fork (Pre-priming complex)
•Single strand binding proteins (SSBPs) bind to separated
strands of DNA and prevents reannealing.
•As unwinding proceeds, DNA gyrase (topoisomerase II)
relaxes supercoiling.
•Primase complexes with helicase, creates RNA primers
(pppAC(N)7-10) on the strands of the open duplex
(Primase+helicase constitute the Primosome).
•DNA pol III holoenzyme comes in and extends the RNA
primer (laying down dNTP's) on the leading strand.
• Pol I removes the RNA primer regions of the Okazaki
fragments via 5' to 3' exonuclease activity.
•Pol I exits and ligase joints the DNA fragments (on lagging
strand).
Rolling-circle replication

(1) by many viruses to duplicate

their genomes, (2) in bacteria
to transfer DNA from donor
cells to recipient cells during
conjugation and (3) in
amphibians to amplify
extrachromosomal DNAs
carrying clusters of ribosomal
RNA genes during oogenesis.
 Eukaryotic DNA polymerases

Six identified* :
(At least 13 different )

Enzyme a** (I) d*** (III) e (II) b g

location -nuclear -nuclear nuclear nuclear mitochondrial

function -priming -elongation repair repair replication
of both of both
strands strands

3’-5’ No Yes Yes No Yes

exonuc.
5’-3’ None; primer removal: Ribonucleases H1 and FEN-1; Pol δ
exonuc. them fills in the gaps

*also ζ pol in repair, **low processivity; adds only a short DNA sequence to the primer,
then polymerase switching, ***main polymerase
 Each eukaryotic chromosome
contains many replicons
• 250-400 in yeast, 25.000 in mammals. Origins in
yeast: Autonomously Replicating Sequences (ARSs)
• Hard to measure size of average replicon since
adjacent ones fuse. Thought to be from 40 to 200
kb.
• No termination signals. Terminate by meeting one
in opposite direction.
• Active replicons are clustered; 20-80 adjacent
replicons are bound to a group of sp. proteins:
Origin Recognition Complex (ORC). 300-500 active
at any given time in mammals.
• If all replicons active, replication could be complete
in 1 hr, but S phase lasts about 6 hr. This implies
that only about 15% are active at any given time.
In eukaryotes, Pol α is required for the
initiation of replication at origins and for
the priming of Okazaki fragments during
the discontinuous synthesis of the lagging
strand. Pol α exists in a stable complex
with DNA primase; indeed, they copurify
during isolation. The primase synthesizes
the RNA primers, which are then
extended with deoxyribonucleotides
by Pol α to produce an RNA–DNA chain
about 30 nucleotides in total length.
These RNA–DNA primer chains are then
extended by Pol δ . Pol completes
the replication of the lagging strand, while
polymerase ε catalyzes the replication of
the leading
strand.
 NOTICE!!
• Smaller replicons (100-200 kb) in
eukaryotes.
• Okazaki fragments: 1000-2000 nts in E.
coli, 100-150 nts in eukaryotes.
• Rate of synthesis: 100 kb/min in E. coli,
20X faster than that in eukaryotes; still it
takes much shorter time to replicate whole
genomic DNA in eukaryotes.
 The telomere problem: The difficulty
encountered during the replication of the
ends of linear chromosomes
 Telomerase
Telomerase:
– is a ribonucleoprotein
– Its single RNA molecule (159 base RNA) provides an AACCCC (in
mammals) template to guide the insertion of TTGGGG
– Its protein component — called hTERT in humans ("human TElomere
Reverse Transcriptase") — provides the catalytic action
– Thus telomerase is a reverse transcriptase; synthesizing DNA from an
RNA template
Aging :
– In most eukaryotic somatic cells, the telomerase activity stops
shortly after the cell differentiates.
– After this, the chromosomes gradually shorten with each
division (telomere length: cellular clock)→cell senescence→cell
death
– Malignant cells maintain telomerase activity: Aging vs
immortality (cancer)
The telomere solution

unorthodox
H bonding
DNA-dependent RNA Polymerase
(rpo A, B, C, D genes)
 The interaction of RNA polymerase with
the promoter: Consensus sequences
Promoters typically consist of a 40 bp region on the 5'-side of the transcription start site

cis-acting elements: The first base is

adjacent parts of always a purine
the same DNA
molecule

(-10 region)

(-35: 35 bases from the

start of transcription; +1)
 Alternative Sigma Factors

e.g. In E. coli: Sigma 28, 32, 54 etc. in

addition to 70.
 Transcription in prokaryotes
binds to ca. 60 bp;
→helix unwound
locally

(α2ββ’) ca. 8 nts

Template Strand vs Partner Strand

-----------
sense strand, or coding strand

antisense strand, or noncoding

strand
 Transcription Initiation
• Step 1 is the binding of RNA pol to the promoter region
• The sigma subunit for selectivity (determines initiation frequency and
transcription direction)
• Once sigma binds the promoter, it forms a closed promoter complex
• Step 2 is the “melting” of the double helix
• Polymerase unwinds about 12 base pairs to form open promoter
complex (from –10 to about +1 producing a single stranded DNA in the
active site of RNA pol
• In Step 3, once the open promoter complex is formed, RNA pol catalyzes
the insertion of the first 5’ ribonucleotide which is complementary to the
1st nucleotide. No primer is required!
• Subsequent complements are inserted and linked together by
phosphodiester bonds
• After several ribonucleotides added, the sigma subunit dissociates and
elongation proceeds
 Elongation and Termination
– Core RNA polymerase adds nucleotides to form
complementary mRNA strand (transcript)
– In E. coli, 50 nucleotides/sec at 37 oC

– Topoisomerases precede and follow polymerase to relieve

supercoiling

– Stop codon does not stop transcription, RNA polymerase will

run over stop codon and keep going

Termination may occur via an intrinsic terminator in the DNA

sequence(ρ-independent) or via a protein rho (ρ-
dependent)
Inverted repeats (Palindromic) in transcribed
DNA lead to formation of a stem-loop
structure in the RNA
 Alternative termination (ρ-dependent): Rho
protein binds to sp. sequences (rut), Rho pulls
RNA pol off RNA
• ρ-dependent
terminators have
inverted repeat in
DNA sequence, but do
not have repeated A’s
• ρ-protein required for
termination; large
hexameric; helicase
(ATP-req.); binds
tightly to RNA
 Prokaryotic mRNAs often contain the coding regions of
two or more genes; such mRNAs are said to be multigenic.Mostly
monogenic in eukaryotes.
 Transcription is more complicated in eukaryotes:
Eukaryotes possess three RNA polymerases: I, II and III (or A, B and C)
1. RNA polymerase I, transcribes major rRNAs; location nucleolus

2. RNA polymerase II, transcribes mRNAs and some snRNAs; location

nucleoplasm

3. RNA polymerase III, transcribes tRNAs, 5S rRNA, and snRNAs; location

nucleoplasm

RNA polymerases IV and V have been identified only in plants; however

they may exist in other eukaryotes, especially fungi. RNA polymerases IV and
V play important roles in turning off the transcription of genes by modifying
the structure of chromosomes, a process called chromatin remodeling. RNA
pol V also synthesizes transcripts that are processed into short RNAs called
small interfering RNAs (siRNAs) that are important regulators of gene
expression.
Transcription is more complicated in
eukaryotes:

– Cis acting elements: TATA box (-25; e.g.

TATAAAA; nonsp; denaturation) and CAAT box (-
80 from start; e.g. GGCCAATCT; increases in
vivo) boxes, Enhancers (location varies; for high
eff. transcription; not engaged in template
binding)
– Transcription Factors (TFs; trans-acting
elements): TFs bind to the DNA to assist in
initiation (Pol II can not bind and initiate in their
absence):, TFIIA, TFIIB, .., etc. Also TATA-binding
proteins (e.g. TFIID; TBPs; pre-initiation complex)
 Prokaryotic vs Eukaryotic
Transcription
Prokaryote
• All promoters upstream of functional gene
• Main promoter consensus sequences TATAAT (-10) and TTGACA (-35)
• One RNA polymerase with σ subunit makes mRNA, tRNA, rRNA
• No enhancers
• mRNA is primary transcript – “ready to go” – short lifetime (just a few
minutes)

Eukaryote
• Promoter positions differ for each polymerase- not all upstream
• Main consensus sequence TATA box (-25) and CAAT box (-60 to -120) Plants
have AGGA instead of CAAT
• RNA POL I – rRNA
• RNA POL II – mRNA
• RNA POL III –5S rRNA, tRNA
• Enhancers to increase transcription
• In eukaryotes, the population of primary transcripts in a nucleus is called
heterogeneous nuclear RNA (hnRNA) because of the large variation
in the sizes of the RNA molecules present. Initial product of transcription
is not usable; primary transcript to be processed. Longer life time
(hours/days)
An interpretive
drawing of an electron
micrograph of a hybrid
molecule: Heteroduplex
Chicken ovalbumin gene
with 7 DNA introns and
mature ovalbumin mRNA
 Post-transcriptional RNA processing in
eukaryotes (from hn RNA/RNP in
nucleus→mature RNA)

Poly(A)
polymerase

Guanylyl- and methyl transferases

 5’- Capping
Capping: Modification of 5’
end of eukaryotic mRNA Phosphorylase

– May play a role in

translation initiation
– May provide increase Guanylyltransferase
mRNA stability (protects
from nuclease attack)
– Transport to cytoplasm Guanine 7-methyl-
transferase
5’-cap
– 7-methylguanosine linked to
the 5’-terminal residue of the
mRNA via a 5’ to 5’ 2’-O-methyl-
triphosphate linkage transferase
 Poly-adenylation
cleavage site
5’-cap
Coding sequence
AAUAAA GU-rich

10 – 30 nts 20 – 40 nts

Endonuclease

AAUAAA
ATP
Polyadenylate polymerase
RNA+ nATP = RNA-(AMP)n + nPPi
PPi
AAUAAA AAA(A)n

80 – 250 A’s
 Polyadenylate polymerase

• Adds 80 – 250 nts to the end of mRNA

• Requires the cleaved mRNA as a primer
• Does NOT require a template for synthesis
• Specific for one nucleotide (ATP)
• Polyadenylation increases mRNA stability
RNA+ nATP = RNA-(AMP)n + nPPi
 Introns

Intervening Sequences or Split Genes

 Classification of introns
• Group I introns
widely distributed in protists, bacteria and
bacteriophages; first discovered in ciliated
protozoan Tetrahymena (1982)

Relevant splicing mechanism:

– Introns are self-splicing (autocatalytic;
ribozymes)
– Does NOT require ATP for splicing
– involves two transesterifications
– 1st transesterification triggered by 3’OH of a
free guanine nucleoside or nucleotide co-
factor (GMP, GDP, GTP)
Splicing of
group I introns

1. The 3’ OH of a free
guanosine attacks the
phosphate at the 5’
splice site resulting in
the 1st trans-
esterification

2. The 3’ OH of the 5’ exon

becomes a nucleophile
The 2nd trans-
esterification results in
joining of exons.
Transesterification reaction
5’ Splice-site: 5’-…U-A…3’
 Classification of introns
• Group II introns
found in fungal and plant mitochondria, algal
plastids, bacteria and Archaea.
Splicing mechanism:
– Introns are self-splicing
– Does NOT require ATP for splicing
– involves two transesterifications
– 1st transesterification triggered by the 2’ OH of
an internal adenylate located within the
intron
– Involves formation of a lariat intermediate
 Splicing of
group II introns
1. The 2’ OH of a specific
adenosine in the intron
attacks the 5’ splice site,
thereby forming a lariat.

2. The 3’ OH of the 5’ exon

triggers the 2nd
transesterification at the 3’
splice site thereby joining
exons together
 Classification of introns
• Third class of introns (non-self splicing):
Spliceosomal introns
– Largest class
– Nuclear mRNA primary transcripts

Splicing mechanism:
– Involves lariat formation similar to group II
– Requires RNA-protein complexes (snRNPs)
= small nuclear ribonucleoproteins (“snurps”)
 Spliceosome
Structure
• 60S dynamic structure (may contain ~ 50 proteins)
• snRNPs
• splicing factors
• assembly of spliceosome requires ATP
Function
• Provide high accuracy of splicing
Splicing defects
• Estimation: 15% of all genetic diseases associated
with mutated splice sites
 snRNAs
Length
snRNA Function
(nts)
Binds 5’ splice site, then 3’ splice
U1 165
site
Binds the branch site and forms
U2 185
part of the catalytic center
U4 116 Masks the catalytic activity of U6

U5 145 Binds the 5’ splice site

U6 106 Catalyzes splicing

Splicing mechanism
5’ splice site (GU) branch site 3’ splice site
(A)

Exon 1 Intron Exon 2

U1
GU U2
A AG
U5
U4
U6 ATP

AG
U1 U2
U5
U4
U6
 Another group of Introns: tRNA
and/or archaeal introns
found in the nuclear tRNA of eukaryotes and
in archaeal tRNA, rRNA and mRNA.

Splicing endoribonuclease (cleaves intron

boundaries at both ends) in both eukaya
and archaea. tRNA ligase subsequently
joins the intron halves to yield a complete
tRNA while circularizing intron (as in the
case of tRNA of eukaryotes ?) .
 Alternative splicing
• generates protein diversity (several hundred thousands
different proteins from 25.000 to 35.000 genes in the
haploid genome)
• allow generation of different splice variants=isoforms
– in different tissues
– at various developmental stages
• Estimates: 30-60% of human genes undergo alternative
splicing
Pre-mRNA

Splice variants:

Number of splice variants (SV)

SV = 2 number of internal exons
 Alternative splicing

An exon may be flanked by regulatory elements on both 5’ and 3’ sites. Depending

on the stage in development or tissue type:
Certain elements are recognized by activator or repressor; stimulating exon
inclusion or exon skip, respectively
 Changes alter what?
• Enzymatic activity,
• Receptor-binding capacity,
• Protein localization in cell
So, very important in development,
apoptosis, several genetic disorders
↓
PROTEOMICS
Understanding the distribution of
splice variants:
• Physiological function of the genes
• Targeting pharmaceuticals (may be
effective against one variant, but not
another)
• Diagnosis (Presence/level may be the
cause/indicative of disease/disorder)
 Two well-studied cases:

Immunoglobulin synthesis in lymphocytes

and
Sex determination in Drosophila

HOMEWORK!
(summaries; each max. 1 page)
 RNA Editing (late 1980s):
Pre-mRNA seq. is changed prior to
translation

1. Substitution Editing

•Editing of mRNA occurs when

adenosine deaminase acting on
RNA (ADAR) acts on an A. A to I

•Also C to U

•In some nuclear-derived

eukaryotic RNAs, also in mt and
cp RNAs of plants.
 RNA Editing (late 1980s):
Pre-mRNA seq. is changed prior to
translation

2. Insertion/Deletion
editing
•Pre-edited RNA base pairs
with a guide RNA on both
sides of the region to be
edited.
•In mt RNA of protozoa (e.g.
Leishmania) and slime molds
(e.g. Physarum)
•The guide RNA is also
transcribed from mt genome;
provides a template for the
insertion of uridines.
•The mRNA produced by the
insertions is complementary to
the guide RNA.
 C to U editosome:
Protein complexes

Editing alter physiological parameters; e.g. response time of receptors

 C to U RNA Editing: APOB
Apolipoprotein B lipid-carrying protein; a component of
the plasma lipoproteins and is crucial for the
transport of cholesterol and of triglycerides in the
plasma to tissues. Occurs in the plasma in 2 main
isoforms, APOB48 and APOB100.; short (intestine)
and long (liver), same gene; site-sp. (at residue 2153)
deamination of C to form U→a glut codon to stop
codon→apoB100 (unedited) to apoB48 (edited;)

• Both are coded by APOB and by a single mRNA

transcript larger than 16 kb. As a result of the RNA
editing, APOB48 and APOB100 share a common N-
terminal sequence, but APOB48 lacks APOB100's C-
terminal LDL receptor binding region.
 C to U RNA Editing: APOB
• In humans, this editing event occurs in the small intestine but not in
the liver.
• The APOB100 (bad cholesterol) isoform is synthesized only in the liver
and used to assemble the very-low-density lipoprotein (VLDL) that is
necessary for the transport of endogenously synthesized
TRIGLYCERIDES and cholesterol to the tissues. VLDL is metabolized
to intermediate-density lipoprotein(IDL) and subsequently to LOW-
DENSITY LIPOPROTEIN (LDL). VLDL, IDL, LDL originate from liver.
APOB "unlocks" the doors to cells and thereby delivers cholesterol to
them..
• (
High levels of APOB LDL cholesterol) is one of the main risk factors
leading to plaques, causing coronary heart disease.
• Conversely, APOB48, which lacks APOB100's C-terminal LDL receptor
binding region is generated in the small intestine and is necessary for
the synthesis and secretion of CHYLOMICRONS (=Large lipoprotein
complex formed in the intestine that transports dietary fats from the
intestine to the liver and to adipose tissue).
 C-to-U RNA editing of apolipoprotein B.The model for an ∼35-nucleotide region of apoB RNA
flanking the edited base (asterisk)

Blanc V , Davidson N O J. Biol. Chem. 2003;278:1395-1398

©2003 by American Society for Biochemistry and Molecular Biology

 High-density lipoprotein (HDL)
particles
Transport cholesterol back from the vessel wall to the liver for excretion
in a process known as reverse cholesterol transport (RCT), i.e.
cholestrol export, thus preventing atherosclerosis Having large
numbers of large HDL particles correlates with better health outcomes

After most of the

lipids in the
chylomicron have
been digested,
APOB48 returns to
the liver as part of
the chylomicron
remnant, where it
is endocytosed and
degraded.
THE GENETIC CODE: The 64
codons and the amino acids

UNIVERSAL,
but...
 Codon Usage Bias (Codon Preference)
Differences in frequency of occurrence of synonymous codons in coding DNA.

A balance between genomic GC content, mutational biases and natural selection for
translational optimization (rate & accuracy)

Greater in highly expressed genes

The Codon Adaptation Index (CAI)is the most widespread technique for
analyzing Codon usage bias by measuring the deviation of a given protein coding
gene sequence with respect to a reference set of genes. Ideally, the reference
set in CAI is composed of highly expressed genes, so that CAI provides an
indication of gene expression level under the assumption that there is
translational selection to optimize gene sequences according to their expression
levels. The rationale for this: highly expressed genes need to compete for
resources (i.e. ribosomes)in fast-growing organisms leading to highly expressed
genes using mostly codons for tRNA species that are abundant in the cell.
Overlapping genes: In some
viruses and mtDNA
Loops contain modified bases
(can not form bp)

Always pGp-5’ at 5’terminus

30-40 different tRNAs in bacteria, 50
in plants and animals:
Isoaccepting tRNAs

3’ACC
 Unusual bases (Rare bases;
odd bases)
Purine hypoxanthine

Formed from posttranscriptional modification

 Degeneracy: The Wobble
Hypothesis
Wobble hypothesis to explain degeneracy of the code
(Francis Crick; 1966):
– The first two nucleotides of codons often determine
the amino acid, with the third position varying

– The third codon position would be subject to relaxed

base-pairing (wobble rules; wobble pairing):

Condon 3rd tRNA

– G can base pair with U in
Position (Anticodon)
tRNA
U A
G C
– Some tRNA nucleotides C or U G
are modified – I can pair A or G U
with A, C or U A, U or C I
 Redundancy in genes coding for rRNAs
(rDNAs): Moderately repetitive DNA
• In E. coli: 7 copies of a single seq. which is
30Senzymatically cleaved to 23S, 16S and 5S

• In eukaryotes; hundreds of copies, e.g. 120 in

Drosophila. In humans: 45S enzymatically
cleaved to 28S, 18S and 5.8S. 5S rDNA is distinct

• Each cluster in eukaryotes: Tandem repeats

separated by noncoding spacer DNA
CHARGING tRNAs
20 diff. aa tRNA
synthetases, regardless
of a greater number of
tRNAs
Mg2+ also required

In bacteria, AGGAGG (upto 6; only

purine; Shine-Dalgarno sequence)
preceding (8 bp upstream) start
codon base pairs with 3’ end of 16S
rRNA of small ribosomal subunit:
facilitates initiation

ACCAUGG (Kozak seq.) in

eukaryotes
RF1, 2 and 3
 A single polypeptide chain folds on itself, several chains
run in either parallel or antiparallel fashion next to one
another. Stabilized by hydrogen bonds formed between
adjacent chains. Somewhat less common than the alpha
Rod-like, has the greatest possible theoretical
stability.
helix.

(Zig-zag plane)
The higher-level assciation of β sheets has been implicated in
(Right-handed formation of the protein aggregates and fibrils observed in
many human diseases, notably the amyloidoses such as
helix; spiral conformation)
Alzheimer's disease.
 (3-D); twists, turns, loops
around itself

Co-linearity
-covalent disufide bonds
-polar, hydrophilic R
groups interact with water
-nonpolar, hydrophobic
R-groups inside the molecule
interact with each other

(Oligomeric protein)
multi-subunit;
e.g. polymerases
 Protein tertiary structure
- folding and packing of secondary structure elements, super-
secondary structure elements, domains
- tertiary folding is stabilized by some non-covalent interactions
- H-bonding
- ionic interactions (salt bridges)
- van der Waals forces
- ‘hydrophobic’ interactions
- can be stabilized by covalent bonds: disulphide linkages
Unstable in cytosol as a
-Cys reducing env., but are
-Cys -S mostly found in extracellular,
secreted and periplasmic
-Cys proteins as well as
Oxidizing -Cys -S for cytosolic proteins under
agent oxidative stress!!!
CystEine Cystine
We are able to predict the secondary structures of proteins from their sequences quite
well (75% accuracy), but fail miserably at predicting the tertiary structures.
4 subunits (each is a protomer;
oligomeric protein
 Most proteins demonstrate a mixture of
these structures
• Fibrous proteins: Elongated, strand-like (filamentous), insoluble
in water, weak acids and weak bases but soluble in strong acids
and alkalis. A single unit or structure is repeated multiple times.
The peptide chains are bound together by strong intermolecular
hydrogen bonds. Highly resistant to digestion by enzymes.

• Globular proteins: Spherical in shape and soluble in water, acids

and bases: β-sheet core, many α-helical areas. Chains change
directions. Held together by weak intermolecular hydrogen
bonds.

**Fibrous proteins generally have only primary and secondary

structure whereas globular proteins have tertiary and
sometimes quaternary structure in addition to primary and
secondary structure.
Fibrous proteins: The secondary structure (either a-helices or β-pleated sheets)
forms the dominant structure. They play a structural or supportive role in the
body, and are also involved in movement (as in muscle and ciliary proteins). e.g.
keratin (found in hair, horns, nails, feathers, etc.; 7 aa. repeating structure),
collagen (major component of our connective tissues; e.g. tendons at a joint for
joint mobility), silk, spider web, etc. All needed for the formation of tough
structures like connective tissue, tendons and fibers of the muscle.
composed only of β-sheets
Globular proteins: Enzymes, transport proteins and receptor proteins.
Hemoglobin, myoglobin, immunoglobins, insulin and milk-protein casein
Internal Protein (Protein's Intron)
Different proteins with diverse functions:
Vacuolar-type ATPase, Cell division, metabolic enzymes, DNA and RNA
polymerases, proteases, ribonucleotide reductases, and more…
 central Homing Endonuclease Region
is found in inteins, so intein-containing proteins
are bifunctional
(can cleave DNA also)

inteins as small
as 134 amino acids
can splice out
of precursor proteins
(monofunctional inteins)
 Inteins as Mobile Genetic Elements: Homing
Endonuclease Activity

If an intein-less allele of the extein gene enters the cell

as the result of conjugation, infection, transformation or any other means,
the intein gene can mobilize into the intein-less extein gene.

Homing endonucleases make double-strand breaks in DNA

at or near the insertion site (home)
of intein-containing gene in host protein alleles
that lack the intein
Gene conversion
involving double-strand break repair mechanisms is initiated
Once the homing endonuclease cleaves the intein-less extein gene,
the only copy of the gene remaining for repair of the DNA break
is the intein-containing gene.

gene conversion from intein-minus to intein-plus is very efficient

Intein mobility (Selfish DNA)
 Homing Endonuclease Function

• Homing endonuclease are site specific, rarecutting

restriction enzymes that recognize along DNA sequence
between 12-40 bps creating a double strand break.

• Most inteins encode a homing endonuclease!!!These enzymes

cleave genes containing similar extein sequences.

• Repair occurs by homologous recombination with the intein

genes. Thus inteins insert themselves into sites in the genes
with similar extein sequences.

• The role of the endonuclease is to enable intein/intron to

horizontally transfer to unoccupied intein/intron integration-
sites via a process termed ‘Homing’.
Protein Domains

• 50-300 aa
• functional capability
• unique folding
 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Differential intron splicing (already covered!)

and exon shuffling explain how one gene can
produce more than one protein

One or more
exons is copied
(duplication;
but part of
genes) and
inserted
elsewhere to
give rise novel
arrangements
of exons (no
need to
reinvent
wheel!)

11-146
 Two proteins that are similar
in certain regions
Tissue plasminogen activator (PLAT)
Coagulation factor XII (F12).

Kringle domains are believed to play a role in binding mediators (e.g., membranes, other proteins or
phospholipids), and in the regulation of proteolytic activity.
Exon Shuffling
• Transposons can drive "exon-shuffling" to
create new genes
 Proteins are the molecular tools
for most cellular functions

TYPE FUNCTION EXAMPLE

Structural proteins Support Collagen, Elastin,
Keratin
Storage proteins Storage of amino acid Ovalbumin,
Casein
Transport proteins Transport of other Hemoglobin
substrate
Hormonal proteins Coordination of and Insulin
organism’s activities
Receptors proteins Response of cell to Receptor in nerve
chemical stimuli transmit route
Contractile proteins Movement Actin, Myosin
Defensive proteins Protecton against Antibodys
disease
Enzymatic proteins Selective acceleraton Trypsin, ATPase,
of chemical reactions GAPDH
Post-translational Processing of Proteins

• Protein folding

• Proteolytic cleavage

• Chemical modification

• Intein splicing
 Post-translational processing and
modification-FOR FUNCTION
Protein processing
Cleavage
• N-terminus methionine entirely or its formyl group is usually removed in prokaryotes.
In eukaryotes, methionine entirely can be removed.
• Preproinsulin to insulin (occurs in cells)
• Pre-prothrombin to prothrombin (in cell) cleaves off leader or signal peptide
• Prothrombin to thrombin (occurs in blood)

Modification
• N-terminus methionine’s amino group is removed or acetylated
• Disulfide bridge (CySH+CySH to CyS-SCy)
• Glycosylation (covalently; e.g. antigens in ABO blood system)
• Methylation or acetylation
• Lipid-linking
• γ-carboxylation
• Phosphorylation (of –OH groups of certain aa.s by kinases ) of e.g. tyrosines, for
ionic bonding with other molecules
• Hydroxyproline (in collagen)
• Complexation with metals (e.g. Hb)
 Protein modifications:
requirements for activity
- cleavage and covalent modifications of proteins (often after synthesis) but
may also be co-translational
I. CLEAVAGE
some proteins require sections of the polypeptide chain
to be removed for correct maturation.

A zymogen is a catalytically inactive protein precursor

that must be cleaved proteolytically to be activated
INSULIN H2N-
Synthesized as PREPROINSULIN

-1st cleavage removes signal sequence (PRE)

- 2nd and 3rd cleavages remove joining (PRO) H2N-

peptide sequences
disulfide
- di-sulphide bonds hold the two peptides H2N- bonds
together H2N-
 Covalent Modifications
Sometimes proteins are covalently modified after synthesis
These modifications can be:
1. Required to obtain the active conformation (e.g.. collagen)
2. Used to control the activity of a protein
(e.g. histones, signal transducing proteins, etc.)

COLLAGEN: Proline  Hydroxyproline

This requires Vitamin C; No Vitamin C  No Hydroxyproline  Scurvy
Due to weakening of collagen fibres- hydroxylation of prolines
somehow stabilizes structure

O
O
C OH
C R

HN
OH
R N
H

OH
 Prothrombin, histones
Prothrombin: Glutamate  gamma-Carboxy Glutamate
This requires Vitamin K; No vitamin K  No Blood Clotting
O
O
H2N CH
CH C OH
H2N CH
CH C OH
CH
CH 2
CH
CH 2
CH 2
CH
CH
CH
C O
O C C O

O-
O- O-

Histones: Histones are proteins involved in the folding/compacting of

nuclear DNA. They are often modified in regions of active transcription.
Acetylation of Lysine is the MOST common.
OH OH

O C O C
H2 H2 H2 H2 H2 H2 H2 H2 H
CH C C C C NH
NH 2
2 CH C C C C N CH
C
3

NH 2 NH 2
O
 Post translational modification
in rough ER and golgi
Post translational modifications in golgi and rough ER
include glycosylation:

the addition of sugar residues to

serine/threonine: O-linked,
asparagine: N-linked
 Molecular Chaperones:
on successive ATP binding and hydrolysis;
having ATPase activities
Chaperonins
• Large, oligomeric, ring-like
• Directly assist folding
• Two subfamilies:
1. Gro EL-Gro ES system (or
Hsp 60); well-studied in E.
coli
2. Thermosome (archaea)
and TRiC (eukarya)
systems
All dependent
Chaperons
• Crucial for maintenance
of native conformation
• Received much attention
recently:
1. Hsp 90
2. Hsp 70 with co-
chaperone Hsp 40
(bacterial homologues
are DnaK and DnaJ,
respectively)
3. Prefoldin (in archaea and
eukarya; does not req.
ATP)
Note: Some archaea (not all)
also contain DNA K and
DNA J
Three mechanisms for aiding
folding

e.g. DnaK

e.g. DnaJ-stimulates
ATP hydrolysis by DnaK
 Nascent
chain-associated complex

Trigger
factor

Prefoldin
 Hsp 60 in mitochondria/GroES-
GroEL complex in bacteria

• GroEL (14 subunits) forms two stacked 7-

membered rings of 60 kD subunits; GroES
is a dome on the top

• Protein bind reversibly many times to the

walls of the donut structure, each time
driven by ATP hydrolysis, eventually
adopting its folded structure, then being
released from the GroES-GroEL complex