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Transcription in Bacteria
Transcription in Bacteria
Transcription in Bacteria
Transcription is the first step of gene expression, in which a
particular segment of DNA is copied into RNA by the enzyme,
RNA polymerase.
If the gene transcribed encodes a protein, the result of
transcription is messenger RNA (mRNA), which then will be used
to create that protein via the process of translation.
Alternatively, the transcribed gene may encode for either noncoding RNA genes (such as microRNA) or ribosomal RNA (rRNA)
or transfer RNA (tRNA), other components of the proteinassembly process, or other ribozymes.
Mechanism of transcription
• RNA polymerase is the enzyme
that catalyzes RNA synthesis.
• Using DNA as a template, RNA
polymerase joins, or polymerizes,
nucleoside triphosphates (NTPs)
by phosphodiester bonds from 5'
to 3'.
• In bacteria there is one type of
RNA polymerase and transcription
and translation are coupled (they
occur within a single cellular
compartment).
• As soon as transcription of the
mRNA begins, ribosomes initiate
protein synthesis.
• The whole process occurs within
minutes.
Transcription in E. coli and in Eucaryotes
Procaryotes
Genes are grouped into operons
Eucaryotes
Genes are not grouped in operons
mRNA may contain transcript of
several genes (poly-cistronic)
each mRNA contains only
transcript of a single gene
(mono-cistronic)
Transcription and translation are coupled.
Transcript is translated already during
transcription.
Transcription and translation are
NOT coupled.
Transcription takes place
in nucleus, translation in cytosol.
Gene regulation takes place by
modification of transcription rate
Gene regulation via transcription
and by RNA-processing,
RNA stability etc.
mRNAs are not processed in prokaryotes
mRNAs are processed in in
eukaryotes (splicing , CAP, poly-A tail)
rRNA and tRNA are processed both in eukaryotes and prokaryotes
Transcription and
translation is coupled in
prokaryotes
Initiation of transcription
Sense strand → nontemplate strand = mRNA
Antisense strand → template strand
As soon the growing
mRNA chain separates
from DNA, ribosomes
attach to it and begin
translation on the 5’ end
of the molecule
following right behind
the RNA polymerase
while it is transcribing
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1
2
How does transcription get started?
RNA polymerase binds to a region on DNA called a PROMOTER
Bacterial promoter structure
Bacterial promoters are not absolutely conserved but they do
have a consensus sequence.
Conserved sequence:
sequence When sequences of DNA have exactly
the same series of nucleotides in a given region.
Consensus sequence:
sequence there is some variation in the sequence
but certain nucleotides are present at high frequency.
Bacterial promoter structure
It is convention to indicate the start of transcription by the number +1 and to
use positive numbers to count farther down the DNA in the direction of
transcription (downstream).
If transcription is proceeding to the right, then the direction to the left is
called upstream with the bases indicated by negative numbers.
For the majority of E. coli genes, the promoter consensus sequence
consists of two hexamer sequences.
Centered at −10 is the consensus sequence TATAAT, which is also
known as the TATA box or Pribnow box. (capital letters indicate bases
found in those positions in more than 50% of promoters analyzed; small
letters = less than 50%).
Bacterial promoter structure
Another region with similar sequences among many promoters is
centered at −35.
The consensus sequence at −35 is TTGACA.
The spacing between the −10 and −35 sequences and the start
point for transcription is important, and deletions or insertions
that change the spacing are deleterious.
How does RNA polymerase recognize and bind to
the promoter?
Structure of bacterial RNA polymerase
Comprised of a core enzyme plus a transcription factor called the sigma factor (σ).
Together they form the complete, fully functional enzyme complex called the
holoenzyme.
Structure of bacterial RNA polymerase
The core enzyme
The core enzyme is the component of the holoenzyme that catalyzes polymerization.
It is 400 kD in size and has five subunits: two copies
of the α-subunit (αI, αII), and one copy each of the
β-, β′-, and ω-subunits.
X-ray crystallographic studies revealed a crab claw
shape. Subunits αI, αII, and ω form the base of the
claw, and subunits β and β′ form the pincers. These
pincers form an internal channel (2.7 nm wide). The
enzyme active site is located at this internal channel
where an essential Mg2+ ion is bound.
The core enzyme has high affinity for most DNA. In the absence of σ,
it can initiate synthesis anywhere on a DNA template in vitro.
The σ factor is responsible for decreasing the nonspecific binding affinity of
the RNA polymerase.
The sequence, structure, and function are evolutionarily conserved from
bacteria to humans.
Structure of bacterial RNA polymerase
Sigma factor
In bacteria there are many different σ factors. In E. coli the most
abundant σ factor is σ70. It has a higher binding affinity for the
RNA polymerase core enzyme than other σ factors.
Most of the σ factors share four regions of amino acid sequence
homology that play a role in recognizing the promoter
These four regions are further divided
into subdomains with specific functions.
In the holoenzyme the globular domain
of the σ factors are spread out across
the face of the crab claw.
Structure of bacterial RNA polymerase
Sigma factor
• σ70 is required for specific binding of RNA polymerase to the promoter of
the majority of genes in E. coli. It stimulates a change in shape of the
internal channel where transcription takes place.
• The −35 and the −10 sequences are necessary for recognition by the σ70
factor
• The −10 sequence is the region of contact for the core enzyme.
• In addition, the −10 sequence is necessary for the initial melting of the
DNA to expose the template strand.
• A domain of the σ70 factor binds to the nontemplate strand of the −10
sequence in a sequence-specific manner that stabilizes the initial
transcription bubble.
The sigma factor, σ70 (MW = 72000)
Fragments 2.1 and 2.2 of σ70 bind strongly to β'.
Adjacent helical segments located in fragments 2.3
and 2.4 are involved in recognition of the -10
region of the promoter. The 2.3 region is required
for melting.
In addition, sequences near the N-terminal (1.1 and
1.2) of σ70 were found to be inhibitory to DNA
binding.
The addition of σ to the polymerase core gives the RNA polymerase holoenzyme recognizing a site at
-10 to form the closed complex. In the holoenzyme form, an additional DNA binding domain of σ, the
region 4.2, become unmasked, and this recognizes a second site at -35, approximately 2 helical turns of
DNA away. If the -35 site is recognized, the holoenzyme melts the region -11 to +3 in the DNA, giving
the open complex, and the bubble is stabilized by the ssDNA binding domain of σ at region 2.3.
The region 2.5 interacts with dsDNA from -11 to –17 (spacer region).
Melting of the transcription bubble admits the template strand to the catalytic site, allowing initiation to
proceed.
Alternative sigma factors respond to general environmental changes
E. Coli sigma factors recognize promoters with different consensus sequences
Sigma70 (rpoD)
(-35)TTGACA (-10)TATAAT
Primary sigma factor, or housekeeping sigma factor.
Sigma54 (rpoN)
(-35)CTGGCAC (-10)TTGCA
Alternative sigma factor involved in transcribing nitrogen-regulated genes (among
others).
Sigma32 (rpoH)
(-35)TNNCNCCCTTGAA (-10)CCCATNT
Heat shock factor involved in activation of genes after heat shock.
SigmaS (rpoS)
intrinsic curvature
(-10)TGNCCATA(C/A)T
Alternative sigma factor transcribing genes of stationary phase of growth.
Note the extended -10 element.
The use of different sigma factors gives E. coli flexibility in responding to different
DNAse I Footprinting
1. Prepare end-labeled DNA.
2. Bind protein.
3. Mild digestion with DNAse I (randomly
cleaves ds DNA on each strand)
4. Separate DNA fragments on denaturing
acrylamide gels.
DNase I footprint performed
on an end-labeled DNA
fragmentFIS
Footprint
Samples in lanes 2-4
had increasing amounts
of the DNA-binding
protein (lambda protein
cII); lane 1 had no
protein.
Partially DNase I digested DNA is subjected to linear PCR
DNA-protein
complex
DNase I
Products of DNase I
digestion are primer
extended by linear PCR
using a 5’ end-labeled
oligonucleotide
Sequencing gel
Gel retardation assay
Gel Shift
Electro Mobility Shift Assay (EMSA)
Band Shift
No protein
add protein
Non-denaturing PAGE
Incubating a purified protein, or a complex mixture
of proteins e.g. nuclear or cell extract, with a 32P
end-labelled DNA fragment containing the putative
protein binding site (from promoter region).
*
*
Reaction products are then analysed on a nondenaturing polyacrylamide gel.
The specificity of the DNA-binding protein for the
putative binding site is established by competition
experiments using DNA fragments or
oligonucleotides containing a binding site for the
protein of interest, or other unrelated DNA
sequences.
Retarded
mobility
due to
protein
binding
Free DNA probe
virB
virF
virG
Bound DNA
EMSA
Free DNA
Evaluating the
Binding Affinity
Primer extension
mRNA
5’
3’
annealing
+
G A T
primer-32P
C
Early-log
37°C 10°C
Mid-log Late-log
37°C 10°C
37°C 10°C
mRNA
5’
primer -32P
3’
-10
+1
reverse transcriptase
+24
mRNA
5’
primer
-32P
3’
+42
cDNA
run on denaturating gel
+77
cspA mRNA
The transcription process
consists of three stages:
stages:
Initiation
Elongation
Termination
Initiation
Initiation of transcription
The RNA polymerase holoenzyme initially binds to the
promoter at nucleotide positions −35 and −10 relative to the
transcription start site (+1) to form a closed promoter
complex.
The term “closed” indicates that the DNA remains doublestranded and the complex is reversible.
Initiation of transcription
The complex then undergoes a structural transition to the “open” form
in which approximately 18 bp around the transcription start site are
melted to expose the template strand of the DNA.
Transcription is aided by negative supercoiling of the promoter region
of some genes. Formation of the open complex is generally irreversible
and transcription initiated in the presence of NTPs.
No primer is required for initiation by RNA polymerase.
Initiation of transcription
During initial transcription, RNA polymerase produces and releases short RNA
transcripts of less then ten ribonucleotides (abortive synthesis) before escaping
the promoter (promotor clearance).
It is not clear why RNA polymerase must undergo this period of abortive initiation
before achieving escape, but a region of the σ factor appear to be involved.
Infact, in this step there is sequential displacement of some domains of σ that
would otherwise act as a barrier to the extension of the nascent RNA as it
emerges from the RNA exit channel.
Elongation
Elongation
After about 9-12 nt of RNA have been synthesized, the initiation complex
enters the elongation stage.
As RNA polymerase moves during elongation, it holds the DNA strands apart,
forming a transcription “bubble.”
The moving polymerase protects a “footprint” of ~30 bp along the DNA against
nuclease digestion.
Within the transcription bubble, one strand of DNA acts as the template for
RNA synthesis by complementary base pairing.
Transcription always proceeds in the 5′→3′ direction.
Proofreading
E. coli RNA polymerase synthesizes RNA with remarkable fidelity in vivo. Its low
error rate may be achieved by two proofreading mechanisms.
Pyrophosphorolytic editing
The RNA polymerase use its active site in a simple back-reaction, to catalyze
the removal of an incorrectly inserted ribonucleotide by reincorporation of Ppi.
Hydrolytic editing
The polymerase backtracks by one or more nucleotides and cleaves the RNA
product, removing the error-containing sequence.
Hydrolytic editing is stimulated by Gre factors, which both enhance hydrolytic
function and serve as elongation stimulating factor.
Gre factors ensure that polymerase elongates efficiently and help overcome
arrest in presence of a mismatch.
Termination
Termination of transcription
The RNA polymerase core enzyme moves down the DNA until a
stop signal or terminator sequence is reached by the RNA
polymerase.
There are two types of terminators recognized,
• Rho-dependent
• Rho-independent terminators
E. coli uses both kinds of transcript terminators.
Termination of transcription
RhoRho
-independent termination
Also called “intrinsic terminators” because they cause termination of transcription in the
absence of any external factors.
Terminator is characterized by a consensus sequence that is an inverted repeat.
Stem-loop structures can form within the mRNA just before the last base
transcribed, by the pairing of complementary bases within the inverted repeat.
The inverted repeat sequence in the mRNA is followed by seven to eight uracilcontaining nucleotides. A hybrid helix of U in the RNA base paired with A in the DNA is
less stable than other complementary base pairs.
This property, combined with formation of the stem loop in the exit channel of RNA
polymerase, is sufficient to cause the enzyme to pause, resulting in transcript release.
Termination of transcription
RhoRho
-dependent termination
Rho-dependent termination is controlled by the ability of the Rho protein
to gain access to the mRNA.
Terminator is an inverted repeat with no simple consensus sequence and
no string of Ts in the nontemplate strand.
Rho is a ring-shaped, hexameric
helicase protein with a distinct RNAbinding domain and an ATP-binding
domain.
Rho binds specifically to a C-rich site
called a Rho utilization (or rut site)
at the 5′ end of the newly formed
RNA, as it emerges from the exit
site of RNA polymerase.
Temporary release of one subunit of the hexamer allows the 3′ segment of the
nascent transcript to enter the central channel of the Rho ring.
Termination of transcription
RhoRho
-dependent termination
In an ATP-dependent process, Rho travels along the RNA, “chasing”
the RNA polymerase. When the polymerase stalls at the terminator stemloop structure, Rho catches up and unwinds the weak DNA–RNA hybrid.
This causes termination of RNA synthesis and release of all the
components.
Bacterial gene
expression