An overview of
protein synthesis
via transcription
and translation
References:
1. Genes VIII, by Lewin,
2004, Oxford.
2. Molecular Biology, by
Weaver, 3rd ed.,2004,
McGraw-Hill.
1
Prokaryotic Gene Expression
Promoter
-35
-10
+1
Terminator
Transcription
(RNA polymerase)
Ribosomebinding site
mRNA
ORF
Start codon
Stop codon
Translation
(Ribosome)
Protein
2
Transcription in Prokaryotic Cells
RNA polymerase
RNA pol
RNA pol
DNA template
Coding strand
Template strand
promoter;
terminator
3
Stages of transcription
Template recognition – Initiation – Elongation - Termination
3’
5’
4
RNA pol in
eubacteria
core: a2bb’
holoenzyme:
core +
s factor
s factor is separated
from the core when
holoenzyme is
subjected to an
anion exchange (e.g.
phosphocellulose)
column
5
RNA
DNA
Yeast RNA
polymerase
6
Functions of s factor
Effect of s factor
Promoter recognition.
Promote tight binding
of holoenzyme to the
promoter.
Loosening nonspecific interaction
between RNA pol
and template.
Stimulates
transcription initiation.
7
s converts a loosely
bound RNA pol in a
closed complex to the
tightly bound pol in
the open promoter
complexes.
Supercoiled DNA is a
better template for
transcription, because
it requires less free
energy for the initial
melting of DNA.
RNA pol-promoter binding
8
9
Template
recognition
1. Forming the closed
promoter complex
2. Forming the open
promoter complex
3. Abortive initiation
Initiation
4. Promoter clearance
10
How RNA
polymerase
gets to the
promoter?
Sliding along DNA
does not occur
11
s factor can
be reused
-Rifampicin
Rif R
+Rifampicin
Rif S
Sigma cycle
12
RNA Pol-Promoter Interaction
DNA region covered by holoenzyme is from -55 to +20; that
covered by core enzyme after loss of s is from -35 to +20.
13
DNA footprinting
14
15
RNA Pol-Promoter Interaction
Methylation Interference
Assay
Bases on either the
template or the nontemplate strand that are
more methylated in the
filtrate than in the filterbound DNA are
presumably important in
polymerase binding to
the promoter.
16
RNA pol-promoter contact
-9 to +3
17
Features of bacterial promoters
-35
lac
TTTACA
-10
18 bp
+1
TATGTT
17 bp
trp
TTGACA
TTAACT
17 bp
lPL
TTGACA
GATACT
17 bp
recA
TTGATA
tacI
TTGACA
TATAAT
9 bp
7 bp
trp
lac
TATGTT
18 bp
Consensus
TTGACA
(16-19 bp)
TATAAT
(5-9 bp)
>90% of transcription start point is a purine
18
How many kinds of s factors
are there in a bacterial cell?
What is the structure of s
factor?
19
20
Structure of s factors
Primary s factors
(e.g. s70 of E. coli;
s43 of B. subtilis )
Alternative s factors
Transcription of
specialized genes
(e.g. s54 of E. coli)
Free s cannot bind to the promoter (The N-terminal region
suppresses the DNA-binding region). Only when it is bound
with the core, upon which its conformation changes, can s
binds the promoter.
21
245 aa deletion
s70
Region 1. Present only in primary s. The 245 aa existing
in s70, but not in s43, may be involved in loosening
binding between RNA pol and non-promoter regions.
Region 2. Most highly conserved.
2.1 and 2.2: hydrophobic; binding to pol core.
2.3: involved in DNA melting.
2.4: a-helix; recognition of -10 box.
Region 3. Helix-turn helix DNA-binding domain.
Region 4.
4.2: helix-turn-helix loop; binding to -35 box.
22
Primary vs. alternative s factors in E. coli
Different s factors recognize promoters with different
consensus sequences
s54 is different from other s factors in:
1. The “-35 box” is located 6 bp upstream of the “-10 box”;
2. Sites that are rather distant from the promoter influence
its activity (recognized by an enhancer-binding protein);
3. The free form can bind to DNA.
23
Sigma-switching model
Temporal
control of
transcription
of B. subtilis
phage SPO1
24
Genetic evidence
Isolation of mutants that are unable to do
transcription switch.
Biochemical evidence
Composition analysis of the RNA pol isolated
from different stages.
Other examples :
Control of transcription during sporulation in B. subtilis
Regulation of glutamine synthetase gene (s54)
Regulation of the E. coli heat shock genes (s32)
Stress-resistance genes turned on in the stationary phase (ss)
25
Elongation
Functions of RNA pol core
1. To unwind and rewind DNA
2. To hold the separated strand of DNA and RNA
3. To catalyze the addition of ribonucleotides to the
growing RNA chain
4. To adjust the difficulties in processing by
cleaving the RNA product and restarting RNA
synthesis (with the assistance of some accessory
factors, e.g., GreA and GreB in E. coli)
26
Recover of RNA
polymerase
from pausing
27
Function of a-subunit
Core enzyme assembly; Promoter recognition;
Interaction with some regulators.
Role of a subunit in
UP element recognition:
1. Addition of UP to the core
promoter increases in vitro
transcription by RNA pol
alone.
2. The 94 C-terminal aa are
required for UP recognition.
28
UP element: an AT-rich sequence which stimulates
transcription of the rrnB gene by a factor of 30.
Fis sites: binding sites for Fis, a transcriptional activator.
29
Function of b and b’-subunit
b-subunit
b’-subunit
Phosphodiester bond formation.
Most basic subunit.
(Confers both the rifampicinand streptolydigin-resistance)
Stabilizing RNA pol-DNA complex
during elongation.
Strongest DNAbinding activity.
Forms salt-resistant
contact with the
DNA template.
Forms both the salt-sensitive and
salt-resistant contact with the
DNA template.
30
Topology of elongation
The strain of unwinding is relaxed by the topoisomerases.
31
Termination
Mechanism
r-independent termination
(intrinsic terminators)
Requires:
a hairpin loop
a string of Ts following
the hairpin.
32
Intrinsic
terminators
Stem of hairpin:
G-C-rich; 7-20 bp
Loop: 5 bp or up
Distance between
hairpin and U-run:
7-9 bp
33
r-dependent termination
Half of E. coli terminators; most
are found in phage genomes
Requires a hairpin loop and
r factor
acts as a homohexamer
each subunit contains
an RNA-binding domain
an ATPase domain.
an RNA helicase (separates
RNA-DNA hybrid)
34
r-dependent terminator
50-90 bases long
C-rich/G poor
35
Polar effect on transcription of the downstream
genes caused by a nonsense mutation
36
Negative control:
Control of
prokaryotic
transcription
Repressor
Inducible genes
Positive control:
v.s.
Constitutive genes
Activator
37
Cofactors of
the regulators:
Common features of the
cofactors:
Repressor
Highly specific
Inducer
Corepressor
Activator
Inducer
Not necessarily interact with the
target enzyme
Gratuitous inducers (e.g., IPTG)
Allosteric control of the regulator
Other means of
activation of regulators:
Other positive control
mechanisms:
Phosphorylation
Substitution of s factors
Oxidation
Antitermination
38
Operon
A group of contiguous, coordinately controlled genes
The lac operon
The first operon discovered
(Jacob and Monod, 1961)
Polycistronic mRNA
394