Transcript Section D - Prokaryotic and Eukaryotic Chromosome Structure

Section K – Transcription in
prokaryotes
Contents
K1 Basic principles of transcription
Transcription: an overview, Initiation, Elongation,
Termination
K2 Escherichia coli RNA polymerase
Escherichia coli RNA polymerase, αSubunit,
βSubunit, β’Subunit, Sigma factor
K3 E. coli σ70 promoter
Promoter sequences, Promoter size, -10 sequence,
-35 sequence, Transcription start site, Promoter
efficiency
K4 Transcription, initiation,
elongation and termination
Promoter binding, DNA unwinding, RNA chain
initiation, RNA chain elongation, RNA chain
termination, Rho-dependent termination
K1 Basic principles of transcription —
Transcription: an overview
• The synthesis of a single-stranded RNA from a
double-stranded DNA template.
• RNA synthesis occurs in the 5’3’direction and
its sequence corresponds to that of the DNA
strand which is known as the sense strand.
•
The template of RNA synthesis is the
antisense strand.
• Necessary components: promoter/template,
RNA polymerase, rNTPs, terminator/template
(-) strand is antisense strand.
(+) strand is sense strand
3‘
5‘
5‘
3‘
• Key:
dsDNA
5’
3’
ssRNA
K1 Basic principles of transcription —
Initiation
(1) Binding of an RNA polymerase to the dsDNA
(2) Slide to find the promoter
Promoter: The sequence of DNA needed for RNA
polymerase to bind to the template and accomplish
the initiation reaction; the5’-side (upstream) of the
coding region; the short conserved sequence
(3) Unwind the DNA helix; For base pairing; Begins at
the promoter site
(4) Synthesis of the RNA strand at the start site
(initiation site), this position called position +1
K1 Basic principles of transcription —
Elongation
• Add ribonucleotides to the 3’-end
• The RNA polymerase extend the growing
RNA chain in the direction of 5’ 3’ （E. coli:
40 nt/sec)
• The enzyme itself moves in 3’ to 5’ along the
antisense DNA strand. The helix is reformed
behind the polymerase.
K1 Basic principles of transcription —
Termination
• The dissociation of the transcription complex
from the template strand and separation of
RNA strand at a specific DNA sequence
known as the terminator.
These sequences often contain self-complementary regions
which can form stem-loop or hairpin structure, some need rho
protein as accessory factor.
K2 Escherichia coli RNA polymerase —
Escherichia coli RNA polymerase
RNA polymerase: synthesis of RNA
strand from DNA template.
• 1. Requires no primer for polymerization
• 2. Requires DNA for activity and is most active with
a double-stranded DNA as template.
•
5’  3’ synthesis,
•
Rate: 40 nt per second at 37oC
• 3. Require Mg2+ for RNA synthesis activity
4. All RNA polymerases lack 3’  5’
exonuclease activity, and one error usually
occurs when 104 to 105 nucleotides are
incorporated.
5. Usually are multisubunit enzyme, but not
always.
6. The cylindrical channel in the enzyme
complex can bind directly to 16 bp of DNA.
The whole polymerase binds over 60 bp.
7. Different from organism to organism
The polymerases of bacteriophage T3 and
T7 are smaller single polypeptide chains, they
synthesize RNA rapidly (200 nt/sec) and
recognize their own promoters which are
different from E. coli promoters
155 KD
36.5 KD
36.5 KD
11 KD
70 KD
151 KD
465kd
Core enzyme（核心酶）: 2 ’
for both initiation & elongation
Holoenzyme（全
酶）: 2 ’
for initiation
K2 Escherichia coli RNA polymerase —αSubunit
• Two identical subunits in the core
enzyme
• Encoded by the rpoA gene
• Required for core protein assembly
• May play a role in promoter recognition
K2 Escherichia coli RNA polymerase —βSubunit
1.
2.
•
•

Encoded by rpoB gene.
The catalytic center of the RNA polymerase
Rifampicin ：bind to the β subunit, and inhibit
transcription initiation. This class of antibiotic does not
inhibit eukaryotic polymerases and has used medically
for treatment of Gram-positive bacteria infections and
tuberculosis Mutation in rpoB gene can result in
rifampicin resistance.
Streptolydigins：Inhibits transcription elongation but
not initiation.
 subunit may contain two domains responsible for
transcription initiation and elongation
K2 Escherichia coli RNA polymerase —β’Subunit
1. Encoded by the rpoC gene .
2. Binds two Zn 2+ ions and may participate in
the catalytic function of the polymerase
• Heparin：binds to the ’ subunit and
inhibits transcription in vitro.
• Heparin competes with DNA for binding to
the polymerase.
 ’ subunit may be responsible for binding
to the template DNA .
K2 Escherichia coli RNA polymerase —
Sigma factor
1. Many prokaryotes contain multiple 
factors to recognize different promoters.
The most common  factor in E. coli is 70.
2. Binding of the  factor converts the core
RNA pol into the holoenzyme.
3. s factor is critical in promoter recognition,
by decreasing the affinity of the core
enzyme for non-specific DNA sites (104)
and increasing the affinity for the
corresponding promoter
• 4. s factor is released from the RNA pol
after initiation (RNA chain is 8-9 nt)
• 5. Less amount of s factor is required
in cells than that of the other subunits
of the RNA pol.
K3 E. coli σ70 promoter —
Promoter sequences
• Different  factors recognize different promoters
• Upstream of the start site of transcription (position
+1), thus the promoter sequences are assigned a
negative number (编号为负数)
• Contains short conserved sequences required for
specific binding of RNA polymerase and
transcription initiation
+1
promoter
DNA
Transcribed region terminator
ATACG
TATGC
Antisense strand
K3 E. coli σ70 promoter —
Promoter size
• Consists of a sequence of between 40 and 60
bp
• -55 to +20: bound by the polymerase
• -20 to +20: tightly associated with the
polymerase and protected from nuclease
digestion by DNaseΙ
• Up to position –40: critical for promoter
function
• -10 and –35 sequence: particularly important
for promoter function
K3 E. coli σ70 promoter —
-10 sequence
• A 6 bp sequence is found in the promoters of many
different E. coli genes which is centered at around
the –10 position (Pribnow, 1975).
• A consensus sequence of TATAAT, the first two
bases(TA) and the final T are most highly conserved
• This hexamer（六聚体） is separated by 5 to 8 bp
from position +1, and the distance is critical
• The –10 sequence is the unwinding sequence.
K3 E. coli σ70 promoter —
-35 sequence
Enhances recognition and interaction
with the polymerase s factor
• A conserved hexamer sequence around position –35
• A consensus sequence of TTGACA, the first three
positions (TTG) are the most conserved among E.
coli promoters.
• Separated by 16-18 bp from the –10 box in 90% of all
promoters
The sequences of five E. coli promoters
K3 E. coli σ70 promoter —
Transcription start site
The sequence around the start site influences initiation
• Purine in 90% of all genes
• G is more common at position +1 than
A
• Often, there are C and T bases on either
side of the start site nucleotide (i.e.
CGT or CAT)
K3 E. coli σ70 promoter —
Promoter efficiency
• There is considerable variation in sequence
between different promoters, and the
transcription efficiency can vary by up to
1000-fold .
• The –35 sequence, -10 sequence, and
sequence around the start sites all influence
initiation efficiency.
• Some promoter require activating factor for
initiation. For example, Lac promoter Plac
requires cAMP receptor protein (CRP)
K4 Transcription, initiation, elongation and
termination —
Promoter binding
• The core enzyme (2 ’) has nonspecific
DNA binding (loose binding, 20000 fold less).
• The  factor enhances the specificity of the
core RNA polymerase (2 ’) for promoter
binding (100x)
• The polymerase finds the promoter –35 and –
10 sequences by sliding along the DNA
extremely rapidly and forming a closed
complex
K4 Transcription, initiation, elongation and
termination —
DNA unwinding
• Necessary to unwind the DNA so that
the antisense strand to become
accessible for base pairing, carried out
by the polymerase.
• The initial unwinding of the DNA
results in formation of an open
complex with the polymerase.
K4 Transcription, initiation, elongation and
termination —
RNA chain initiation
• No primer is needed
• Start with a GTP (more common) or ATP
• Initially incorporates first 2 nucleotides.
The first 9 nt are incorporated without
polymerase movement along the DNA
or σfactor release
K4 Transcription, initiation, elongation and
termination —
RNA chain elongation
• σFactor is released to form a ternary
complex of the pol-DNA-RNA (newly
synthesized), causing the polymerase to
progress along the DNA (promoter
clearance).
•
Transcription bubble (unwound DNA
region, ~ 17 bp) moves along the DNA with
RNA polymerase which unwinds DNA at the
front and rewinds it at the rear.
K4 Transcription, initiation, elongation and
termination —
RNA chain termination
• Termination:
Dissociation of RNA
Re-annealing of DNA
Release of RNA pol
Terminator sequence :
• RNA hairpin very common
• Accessory rho protein（辅助ρ蛋白）
K4 Transcription, initiation, elongation and
termination —
Rho-dependent termination
• Some genes contain terminator sequences
requiring an accessory factor, the rho protein
(ρ) to mediated transcription termination.
• Rho binds to specific sites in the singlestranded RNA.
• Rho protein (hexameric protein) binds to
certain RNA structure (72bp)
• Rho hydrolyses ATP and moves along the
nascent RNA towards the transcription
complex then enables the polymerase to
terminate transcription.
Binding
Unwinding
Initiation
Elongation
Termination
RNA polymerase/transcription
and DNA polymerase/replication
RNA pol
DNA pol
Template
dsDNA is better ss/dsDNA
Require primer
No
Yes
Initiation
Promoter
Origin
Elongation
40 nt/ sec
900 nt /sec
Terminator
Synthesized
RNA
Template
DNA
Multiple choice questions
1. Which two of the following statements about transcription are correct?
A RNA synthesis occurs in the 3' to 5' direction.
B the RNA polymerase enzyme moves along the sense strand of the DNA in a
5' to 3' direction.
C the RNA polymerase enzyme moves along the template strand of the DNA in
a 5' to 3' direction.
D the transcribed RNA is complementary to the template strand.
E the RNA polymerase adds ribonucleotides to the 5' end of the growing RNA
chain.
F the RNA polymerase adds deoxyribonucleotides to the 3' end of the growing
RNA chain.
2. Which one of the following statements about E. coli RNA polymerase
is false?
A the holoenzyme includes the sigma factor.
B the core enzyme includes the sigma factor.
C it requires Mg2+ for its activity.
D it requires Zn2+ for its activity.
3.
A
B
C
D
E
F
4.
A
B
C
D
E
Which one of the following statements is incorrect?
there are two α subunits in the E. coli RNA polymerase.
there is one β subunit in the E. coli RNA polymerase.
E. coli has one sigma factor.
the β subunit of E. coli RNA polymerase is inhibited by rifampicin.
the streptolydigins inhibit transcription elongation.
heparin is a polyanion, which binds to the β’ subunit.
Which one of the following statements about transcription in E. coli is
true?
the -10 sequence is always exactly 10 bp upstream from the transcription
start site.
the initiating nucleotide is always a G.
the intervening sequence between the -35 and -10 sequences is conserved.
the sequence of the DNA after the site of transcription initiation is not
important for transcription efficiency.
the distance between the -35 and -10 sequences is critical for transcription
efficiency.
5. Which one of the following statements
about transcription in E. coli is true?
A loose binding of the RNA polymerase core
enzyme to DNA is non-specific and unstable.
B sigma factor dramatically increases the relative
affinity of the enzyme for correct promoter sites.
C almost all RNA start sites consist of a purine
residue, with A being more common than G.
D all promoters are inhibited by negative
supercoiling.
E terminators are often A-U hairpin structures.
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