Replication, Transcription and

Translation
 Central Dogma
Reverse Transcription
DNA Replication

Transcription
Images removed due to
copyright restrictions.

RNA

Translation

Protein
 Flow of information

replication
DNA → DNA
transcription ↓
RNA
translation ↓
protein
5' end ring numbering
system for
-P-O-C deoxyribose
5’
-C
O O 4’ 1’
P
O- O 3’ 2’
C

3’ end ssDNA
HO
In a nucleotide, e.g., adenosine monophosphate (AMP), the base is
bonded to a ribose sugar, which has a phosphate in ester linkage to
the 5' hydroxyl.

NH2 NH2 NH2

adenine
N N N
N N N

N N N N N N
H −2
HO O3P O
5' CH2 CH2
O O
4' H H 1' H H
ribose
H 3' 2'H H H
OH OH OH OH
adenine adenosine adenosine monophosphate (AMP)
Nucleic acids have a NH2
backbone of adenine
N
alternating Pi & N

ribose moieties. N NH2

5' end O − N
Phosphodiester cytosine
− 5'
O P O CH2 N
linkages form as the O
O 4' H H 1'
5' phosphate of one ribose
N O
H 3' 2'H
nucleotide forms an O OH
ester link with the 3' −
O P O
5'
CH2
O
OH of the adjacent O H H
ribose
nucleotide. H 3' H
O OH
−
O P O (etc)
nucleic acid 3' end
O
 H
N H O N
Guanine
Cytosine
N H N N
N N
O H N Backbone
Backbone
Hydrogen H
bond

H
CH3 O H N
N
Thymine N H N N Adenine
N N
Hydrogen
O bond Backbone
Backbone

Figure by MIT OCW.

 Diagram of genetic structure removed due to copyright restrictions.
See Figure 7-4 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms. 11th ed.
Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Replication
 DNA Replication

• A fundamental process

• Experimentally demonstrated
DNA

DNA
DNA
DNA Replication

THREE HYPOTHESES FOR DNA REPLICATION

 Stable isoptopes in biology

SEPARATION OF DNAS BY CESIUM CHLORIDE DENSITY GRADIENT CENTRIFUGATION

(a) Photo of DNA in ultracentrifuge rotor made with UV light
(b) Densitometric trace of UV scan
 15 15
D= N labeled 1. Grow E coli so DNA uniformly N labeled
14 14
L= N labeled 2. Add N labeled to growth media and observe result
over several generations of growth

PREDICTED DENSITIES OF
NEWLY REPLICATED DNA
MOLECULES ACCORDING
TO THE THREE HYPOTHESES
ABOUT DNA REPLICATION
 RESULTS OF CsCl GRADIENT
ULTRACENTRIFUGATION
Image of experimental results removed due to copyright restrictions.
EXPERIMENT SHOWING
See Meselson, and Stahl. "The Replication of DNA in Escherichia coli." DISTRIBUTION OF DNA
PNAS 44 (1958): 674, f. 4. DENSITY IN E. coli CELLS
AFTER 0 TO 4.1
GENERATIONS OF GROWTH

THIS EXPERIMENT ESTABLISHED

THAT DNA REPLICATION IS
SEMICONSERVATIVE
 Conclusion

1. DNA replication is semi-conservative

 DNA Replication Process

DNA

DNA
DNA
Diagram removed due to copyright restrictions.
See Figure 7-12 in Madigan, Michael, and John Martinko. Brock
Biology of Microorganisms. 11th ed. Upper Saddle River,
NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
All DNA polymerases require a primer
DNA is synthesized 5' to 3'

RNA primer DNA

PPP-5' 3'-OH
3' 5'
DNA

Figure by MIT OCW.

PRIMING OF DNA SYNTHESIS BY

SHORT SEQUENCES OF RNA (RED)

DNA POLYMERASE USES THE

PRIMERS AS STARTING POINTS
TO SYNTHESIZE PROGENY
DNA STRANDS (GREEN ARROWS)
Table of the major enzymes involved in DNA replication in bacteria removed due to copyright restrictions.
See Table 7-3 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
 Helicase & topoisomerase
Unwind & remove supercoils in duplex DNA
 ssDNA binding protein
binds to and stabilizes ssDNA

prevents base pairing

ssDNA binding protein

primase
synthesizes a short RNA primer
using a DNA template

primase

RNA primer
(a short starting sequence
made of RNA)
 DNA polymerase III
Synthesizes DNA 5’->3’, by
priming off the RNA primer
on the lagging strand template.

Also has 3’->5’ proofreading activity

DNA polymerase I

Synthesizes DNA from a DNA

template and also
removes RNA primers from the
“Okazaki fragments”.
DNA ligase
Joins DNA strands together by
forming phosphodiester bonds

DNA ligase
replication fork
5'
lagging strand
3'

5'
leading strand

template strands
3'
Leading strand 5'
synthesis

RNA primer

helicase
ssDNA binding proteins
3'
 5'

DNA polymerase

helicase
ssDNA binding proteins
3'
Leading strand synthesis 5'

DNA pol III

helicase
DNA
ssDNA binding proteins 3'
 Proofreading
Pol III removes misincorporated bases using 3' to 5'
exonuclease activity
This decreases the error rate to about 10-10 per base
pair inserted

Diagram of DNA proofreading removed due to copyright restrictions.

See Figure 7-20 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Lagging strand synthesis
(discontinuous) Okazaki
fragment
3' (~1000 bases)
pol III
(primase)
5'
helicase
ssDNA binding proteins
3'
 Primer removal
3' pol III

5'
pol I

5’ to 3’
pol I exonuclease
activity
Ligation

DNA ligase
 5'

RNA primer

Lagging strand

Primase
3' Single-strand binding protein
5'
DNA polymerase III
Helicase

Leading strand
Free 3'-OH

RNA primer

5'
3'

Figure by MIT OCW.

DNA SYNTHESIS HAPPEN BIDIRECTIONALLY, FROM INITIATION SITE

“REPLICATION BUBBLE”
Origin of replication

Newly synthesized
Replication forks DNA
Theta
Structure

Figure by MIT OCW.

 Flow of information
replication
DNA → DNA
transcription ↓
RNA
translation ↓
protein
 Regulatory pathways in prokaryotes

Regulate enzyme synthesis

Regulate
enzyme activity
Substrate Product At translation

No Product At transcription

Enzyme A Enzyme B No Enzyme

Translation
No mRNA
Transcription

Gene A Gene B Gene C Gene D

Figure by MIT OCW.

Prokaryotic transcription
Transcribed regions
RNA polymerase
Promoters
Terminators
Sigma factor
5' end
-P-O-C

O HOO
P URACIL

O- O
C

3’ end ssDNA
HO HO
Transcription

Diagram of RNA transcription removed due to copyright restrictions.

See Figure 7-29a in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
DNA dependent RNA Polymerase (RNAP)
recognizes promoter sequence and initiates
transcription

Initiate Terminate
Promoter region
{
5' 3'

3' 5'

{
1 Gene

Sigma aids in recognition of promoter

and initiation site

5' 3'

3' 5'

Sigma factor
RNA polymerase
(core enzyme)

Figure by MIT OCW.

Synthesis of the mRNA transcript (5’3’)
complementary to one of the twp strands – the
template strand – sigma dissociates

Elongation Phase
Sigma

5' 3'

3' 5' 5'

Transcription begins;
sigma released

5' 3'

3' 5'
RNA chain growth 5'

Figure by MIT OCW.

Elongation phase continues until the RNAP reaches a
terminator and dissociates

5' 3'

3' 5'
Termination site reached;
chain growth stops

5'

5' 3'

3' 5'
3'
Release of polymerase
and RNA
5'

Figure by MIT OCW.

Initiation of transcription begins with promoter binding by
RNAP holoenzyme
holoenzyme = RNAP core + Sigma

Diagram of RNA polymerase and transcription removed due to copyright restrictions.

 Architecture of a vegetative (σ70) promoter

-core promoter recognized by sigma factor

T T G A C A T A T A A T
69 79 61 56 54 54 77 76 60 61 56 82
% occurence 17 bp spacer (43)
A
47
-60
UP Element -35 -10 +1 DSR

Core Promoter
Alternative sigma factors bind to core RNA pol and direct it to
different promoters.

E. coli RNA pol holoenzyme is α2ββ’σ

Sigma 70 is used for ‘normal’ promoters

Sigma 32 is used for heat-shock promoters

Sigma 54 is used for N limitation promoters

Gene Sigma factor -35 Spacing –10

rpoD σ70 TTGACA 16-18 bp TATAAT

rpoH σ32 CCCTTGAA 13-15 bp CCCGATNT
rpoN σ54 CTGGNA 6 bp TTGCA
What dictates the transcriptional activity of a gene?

Promoter strength

How similar are the promoter core elements (-10,-35, and

their spacing) to the consensus?
- in general the more similar they are, the more active
the promoter will be to initiate transcription
- however, some positions are more important
than others
TTGACA ---17bp---TATAAT---A Consensus

TCGACA---17bp---TATTAT---A Strong promoter

TCAGTT---19bp---GATAAC---A Weaker promoter

Non-core sequences can affect promoter strength
1. Extended –10 sequences

some promoters have longer –10 elements

2. UP elements

other promoters have AT rich sequences just upstream of the

–35 that elevate transcription rate

3. Downstream elements

sequences immediately downstream of the start site can

affect the overall efficiency of transcription initiation
 Initiation complexes through elongation

Diagram removed due to copyright restrictions.

Karp, 1999, Molecular Cell Biology, Wiley and Sons

RNAP

+
RNAP RNAP RNAP
Closed complex Open complex Elongation complex
promoter
The transcription cycle

- can be viewed as a
cycle

1. Initiation
Diagram removed due to copyright restrictions.

2. Elongation

3. Termination

Mooney and Landik, 1999. Cell

98: 687
 Mechanism of transcriptional initiation
NTPs
k1 k2 k3 k4
R+P RPC RPO RPI RPE
k-1 k-2 k-3
abortive
Described by a transcripts
equilibrium constant
This transition
called KI
is called
“promoter
KI = RPC/(R + P)
clearance”
described by kIV
R – RNAP The rate of open
P – Promoter complex formation is
RPC – closed complex
often called kII
RPO – open complex
RPI – initiation complex
RPE- elongation complex
 Transcription termination

Diagram of transcription termination removed due to copyright restrictions.

See Figure 7-32 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms. 11th ed.
Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Some differences between eukaryotic &
prokaryotic transcription.
Eukaryotic mRNAs are usually
spliced,capped and tailed, in the nucleus.
Eukaryotes do NOT have classical operons.
RNA polymerase structure/function differ
Initiation complexes differ Sigma factor vs. TBP
Prokaryotic genes very very rarely have introns
 DRUGS THAT INHIBIT TRANSCRIPTION &/or DNA REPLICATION
ANTIBIOTIC TARGET; MODE OF ACTION
Actinomycin D Transcription; inhibits DNA-dependent RNA synthesis by binding DNA

Adriamycin.HCl DNA replication & Transcription; Inhibits DNA and RNA synthesis by binding DNA

Aphidicolin DNA replication ; Inhibits alpha-type polymerase (eukaryotic and viral)

Bleomycin.sulfate DNA replication ; reacts with DNA and causes chain break

Chromomycin A3 Transcription ; Inhibitor of DNA-dependent RNA-synthesis

Mithramycin A Transcription ; inhibits RNA synthesis by complexing with DNA

Mitomycin C DNA replication ; Anti-tumor antibiotic. Binds covalently to DNA

Nalidixic acid DNA replication; Inhibitor of bacterial DNA gyrase (a topisomerase inhibitor)

Netropsin. DNA replication; Peptide antibiotic. Binds to AT-rich regions in the minor groove of DNA

Novobiocin DNA replication; Inhibitor of bacterial DNA gyrase

Rifampicin Transcription ; Inhibitor of DNA-dependent RNA-polymeras

Novobiocin. DNA replication; Causes DNA methylation and DNA strand breaks
 Translation
Coupled transcription/translation Compartmentalization/transcript processing

Diagram of transcription and translation in prokaryotes vs. eukaryotes removed due to coypyright restrictions.
Coupled transcription/translation

Microscopic photographs of transcription and translation removed due to copyright restrictions.

 Flow of information
replication
DNA → DNA
transcription ↓
RNA
translation ↓
protein
Overview of prokaryotic translation
Protein synthesis from an mRNA template.
translated region

mRNA
phe

translation

protein of specific amino acid sequence

Key components of translation
Messenger RNA
Transfer RNA
ribosomes and rRNA
 Simple structure of a prokaryotic gene

σ70-type Promoter
Transcriptional terminator
TTGACA ---17bp---TATAAT---A UUUUUUU

Shine-Delgarno
GGAGGA
ATG Stop

Regulatory
sequences coding sequence
1-500 nts 2-8 nts variable
(~ 5 nts)

Ribosome Binding Site (RBS)

Shine-Dalgarno sequence
~AGGAGG, ribosome binding sequence,
critical for ribosome binding
Start codons
AUG, GUG, or UUG
Stop codons (nonsense codons)
UAA, UGA, or UAG
 Secondary Structure: small subunit ribosomal RNA

5’mRNA AGGAGGU 3’
3’rRNA UCCUCCA 5’

Shine Delgarno seq.

Blue = Universal sites

Courtesy of the Comparative RNA Web Site. Used with permission.

 THE GENETIC CODE
• Series of codons that determines
the amino acid sequence of the
encoded protein.
• Coding sequences have an average
of about 300 codons.
• Except for the stop codon, each
codon specifies a particular amino
acid.
 The genetic code

Table of the genetic code removed due to copyright restrictions.

See Table 7-5 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms. 11th ed.
Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
The genetic code is degenerate.
more than one codon can code
for the same amino acid

UUU → phenylalanine
UUC → phenylalanine
 Synonyms
Different codons can code for the same amino acid

UUU → phenylalanine
UUC → phenylalanine
Not all synonyms are used with equal
frequency. This is called "codon usage
bias".
Codon families

any nucleotide in
the 3rd positions
CUU
CUC leucine
CUA
CUG
Codon pairs any pyrimidine in
the 3rd position
UUU
phenylalanine
UUC
CAA
glutamine
CAG
any purine in
the 3rd position
Codons consist of 3 bases
start
codon
1 2 3 4
codons AUGCAUUGUUCU...

protein fMet - His - Cys - Ser ...

1 2 3 4
 Reading frames

TTC TCA TGT TTG ACA GCT

RF1 Phe Ser Cys Leu Thr Ala>
RF2 Ser His Val *** Gln Leu>
RF3 Leu Met Phe Asp Ser>
AAG AGT ACA AAC TGT CGA
RF4 <Glu *** Thr Gln Cys Ser
RF5 <Glu His Lys Val Ala
RF6 <Arg Met Asn Ser Leu
Structures of amino acids commonly
found in proteins (20).
Selenocysteine – the 21st amino acid
Selenocysteine appears in a number of oxidoreductase
H enzymes e. g. formate dehydrogenase, glycine reductase.
UGA codon, normally nonsense !
Se
(surrounding context allows to serve as ‘sense’ codon)

CH 2
H C NH2
N
COOH
O CH 3
NH

Pyrrolysine – the 22nd amino acid

UAG codon, normally nonsense !
H
Pyrrolysine is found in enzymes involved in methanogenesis C
in a few bacteria and archaebacteria. H2N COOH
Key components of translation
Messenger RNA
Transfer RNA
ribosomes and rRNA
 Diagrams removed due to copyright restrictions.
See Figures 7-36 and 7-34 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Wobble base pairing UUU phenylalanine
UUC

U-G and G-U base pairs are allowed in

the 3rd position of the codon.
codon (mRNA)
5' UUU 3'
3' AAG 5'

anticodon (tRNA)
tRNA charging (adding amino acid)
O
3' 3'
H2N-CH-C-O
R

tRNA aminoacyl-tRNA
(uncharged) (charged)
tRNA charging uses the energy of ATP
 O O O O
H
R C C O− −
O P O P O P O CH2 Adenine
O
NH3+ O− O− O− H H
Amino acid ATP H H
1 OH OH

O O
H
R C C O P O CH2 Adenine
O
− H H + PPi
NH2 O
Aminoacyl-AMP H H
OH OH

Aminoacyl-tRNA Synthetases catalyze linkage of the

appropriate amino acid to each tRNA. The reaction occurs in two
steps.
In step 1, an O atom of the amino acid α-carboxyl attacks the P
atom of the initial phosphate of ATP.
 O O
H
R C C O P O CH2 Adenine
O
NH2 O− H H

Aminoacyl-AMP H H
OH OH
tRNA
In step 2, the 2' 2
tRNA AMP
or 3' OH of the
O
terminal
Adenine
adenosine of O P O CH2
O
tRNA attacks the H H (terminal 3’nucleotide
O−
of appropriate tRNA)
amino acid H
O
3’ 2’
OH
H
carbonyl C atom.
C O
Aminoacyl-tRNA
HC R

NH3+
 Aminoacyl-tRNA Synthetase

Summary of the 2-step reaction:

1. amino acid + ATP • aminoacyl-AMP + PPi
2. aminoacyl-AMP + tRNA • aminoacyl-tRNA + AMP
The 2-step reaction is spontaneous overall, because the
concentration of PPi is kept low by its hydrolysis, catalyzed by
Pyrophosphatase.
There is a different Aminoacyl-tRNA Synthetase (aaRS) for each amino
acid.
Each aaRS recognizes its particular amino acid and the tRNAs coding for
that amino acid.
Accurate translation of the genetic code depends on attachment of each
amino acid to an appropriate tRNA.
Domains of tRNA recognized by an
aaRS are called identity elements.
Most identity elements are in the
acceptor stem & anticodon loop.
Aminoacyl-tRNA Synthetases arose anticodon loop
early in evolution. The earliest
aaRSs probably recognized tRNAs
only by their acceptor stems.
acceptor
tRNA stem
Key components of translation
Messenger RNA
Transfer RNA
ribosomes and rRNA
 Structure of the E. coli Ribosome

Diagram of the structure of the E. coli ribosome removed due to copyright restrictions.

The cutaway view at right shows positions of tRNA (P, E sites) &
mRNA (as orange beads).
 Table of ribosome structure removed due to copyright restrictions.
See Table 7-6 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
 DRUGS THAT INHIBIT TRANSLATION

Inhibitor Comments
Chloramphenicol inhibits prokaryotic peptidyl transferase

Streptomycin inhibits prokaryotic peptide chain initiation, also induces mRNA misreading

Tetracycline inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small subunit

Neomycin similar in activity to streptomycin

BACTERIAL

Erythromycin inhibits prokaryotic translocation through the ribosome large subunit

Fusidic acid similar to erythromycin only by preventing EF-G from dissociating from large subunit

Puromycin resembles an aminoacyl-tRNA, interferes with peptide transfer resulting in premature

termination in both prokaryotes and eukaryotes
EUKARYOTE

Diptheria toxin catalyzes ADP-ribosylation of and inactivation of eEF-2

Ricin found in castor beans, catalyzes cleavage of the eukaryotic large subunit rRNA

Cycloheximide inhibits eukaryotic peptidyltransferase

Prokaryotic 70S ribosome

23s rRNA
5s rRNA 50s
34 proteins subunit

16s RNA 30s

21 proteins subunit
 Secondary Structure: small subunit ribosomal RNA

5’mRNA AGGAGGU 3’
3’rRNA UCCUCCA 5’

Shine Delgarno seq.

Blue = Universal sites

Courtesy of the Comparative RNA Web Site. Used with permission.

 Diagram removed due to copyright restrictions.
See Figure 7-38 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
 NH3+
Growing protein chain

1
NH3+

2
3
NH+3 8 Amino acid residue

4
OH 5
6 7

Transfer RNA

5' 3'
Messenger RNA Ribosome

Direction of ribosome
movement on mRNA

Figure by MIT OCW.

 Diagram removed due to copyright restrictions.
See Figure 7-39 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
RIBOZYMES ARE CATALYTIC RNAS

EXAMPLES :

Rnase P - (cleaves t-RNA presursor -> tRNA

Self splicing introns in eukaryotes

Ribosomes !!!!
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Shailendra Sharma