Transcription in
Prokaryotes
Transcription
Transcription: production
of mRNA copy of the
DNA gene.
Eukaryote model
Transcription
RNA
Not all RNA is translated into protein:
• Some RNA is structural - e.g. ribosomal RNA (rRNA)
• Some RNA is functional - e.g. transfer RNA (tRNA)
• Some RNA is chromosomal (some viruses)
The production of protein-encoding RNA in bacteria
is the subject of this lecture.
Transcription
From which DNA strand is RNA synthesized?
 Transcription usually takes place on only ONE of the
DNA strands (though not necessarily the same strand
throughout the entire chromosome).
Transcription
RNA growth always in the 5'  3' direction
5'-GTCACCCATGGAGG-3' Nontemplate strand
3'-CAGTGGGTACCTCC-5' Template strand
5'-GUCACCCAUGGAGG-3' mRNA
3' mRNA 5'
5'
3'
5'mRNA 3'
5'mRNA 3' 5'mRNA 3'
3' DNA
5' DNA
Transcription
RNA Polymerase
The synthesis of RNA from a DNA template is
carried out by enzymes known formally as DNAdependent RNA polymerases, now simply
referred to as RNA polymerases
Transcription
ACCCATGG
C
A
5'-GT
GG-3' Nontemplate
“CODING”strand
CAUG CC-5‘ Template strand
3'-CA
C
G
T
C
TGGGTACC
A
5'-GUC
RNA polymerase
RNA Polymerase
RNA polymerases have the following properties:
 The enzymes are template dependent, requiring
double-stranded DNA
 The enzymes require the four nucleoside triphosphates
(ATP, GTP, CTP, and UTP)
 The enzymes copy (read) the template DNA strand in
the 3' to 5' direction
 The enzymes synthesize the RNA in the 5' to 3'
direction
Transcription
Order of events in Transcription
1) Binding of polymerases to the initiation site, the promoter.
Prokaryotic polymerases can recognise the promoter and bind to
it directly.
2) Unwinding (melting) of the DNA double helix by a helicase. In
prokaryotes the polymerase has the helicase activity.
3) Synthesis of RNA based on the sequence of the DNA template
strand, using nucleoside triphosphates (NTPs) to construct
RNA.
4) Termination of synthesis. NOTE: the “STOP” codon in the
genetic code for the end of peptide synthesis is NOT the end of
termination.
RNA Polymerase
Prokaryotic RNA Polymerase: Core Enzyme



Chain initiation and interaction
with regulatory proteins
Catalytic center: chain initiation
and elongation
DNA binding
RNA Polymerase

The core enzyme has the ability to
synthesize RNA, however, the initiation
point of RNA synthesis is non-specific.

An additional subunit, the sigma factor,
is required to initiate RNA synthesis at
specific locations in the DNA, termed
the promoter.
RNA Polymerase
Prokaryotic RNA Polymerase: Holoenzyme
 Promoter recognition
Promoters
•
For any given gene, RNA synthesis always starts
at the same point on the DNA, the promoter.
What is a promoter?
•
Hypothesis: Because one RNA polymerase copies
every gene and binds to the promoter in each
gene to do so, the promoters in different genes
must have similarities. Similarities in DNA must
lie in the sequence of nucleotides so the
promoters of every gene must have the same
sequence of nucleotides.
Promoters
David Pribnow tested this
by comparing the sequences
in the promoter regions of
five genes from E. coli. He
found a conserved sequence
of nucleotides in each. This
was called the Pribnow box.
Pribnow
Promoters
• The Pribnow box lies 10 nucleotides from the
transcription start point (TSP). A second was
later found 80 nucleotides away.
-80
TTGACA
-10
TSP
TATAAT
5' DNA
3'
3'
5'
Pribnow box
RNA
Promoters
Consensus sequences
• The sequences found in promoters are to
some extent imaginary. Very few genes
actually contain these sequences but they
all contain a sequence that is only a few
nucleotides different. The consensus
sequence is a “best average”.
Promoters
GCGTTGTCATGC
AATGTGACAGCT
TGCTAGACACAG
GAATTGAGAAAA
CTTTTCACATTC
AGCTAGACAGGG
TCGTTGGCACCA
CCAATGACCATT
ATGTTGACTTGC
TTGACA
gene1
gene2
gene3
gene4
gene5
gene6
gene7
gene8
gene9
consensus not actually
in any of the genes
Promoters
• Just because consensus sequences have
been found, this doesn’t mean that they
are functional. What is the evidence that
they actually work?
Promoters
• Although sequences can vary from the
consensus, some mutations stop the
promoter from working. In these cases, it
demonstrates that the consensus sequence
is a functional promoter.
• Genes that are transcribed strongly have
sequences more like the ideal consensus
than genes that are transcribed weakly.
Transcription
RNA polymerase scans DNA double helix,
searching for a promoter site.
Promoter region in DNA
RNA polymerase
Initiation
(1) Sigma binds to promoter region.
Sigma residues Y425, Y430 and W434
directly involved in the unwinding
(melting) of the double helix.
Initiation
(1) Sigma binds to promoter region,
recognizing both the -35 and -10 regions.
The resulting structure is termed a closed
promoter complex.
The promoter is rich in A and T.
The AT pair involves two hydrogen bonds
whereas the CG pair involves three
hydrogen bonds. Therefore, AT pairs are
easier to separate.
Initiation
(2) After the DNA strands have been
separated at the promoter region by the
helicase activity of the sigma subunit,
forming an open promoter complex. The
core subunit () can then start to
synthesize RNA.
(3) Following initiation, the sigma subunit is
released after approx. 10 ribonucleotides
have been polymerized,
Elongation
Synthesis of the RNA strand
continues until the core polymerase
reaches the termination site.
Termination
In prokaryotes, the transcription is
terminated by two major mechanisms:
Rho-independent (intrinsic) and
Rho-dependent.
Termination
Rho-independent
The Rho-independent termination signal is a
stretch of 30-40 bp sequence, consisting of
many GC residues followed by a series of T
("U" in the transcribed RNA).
The resulting RNA transcript will form a
stem-loop structure to terminate transcription
Termination
The terminator has the
following structure:
Complementary
GC rich
GC rich PolyA
GC rich
GC rich PolyU
DNA
RNA
Termination
stem-loop structure
RNA
C
U C
U
G
G C
A U
C G
C G
GC rich regions C G
G C
C G Poly U
C G
UAAUCCCACAG CAUUUU
Termination
5
TAG
GC rich 1 GC rich 2 ATrich
5
3
As transcription proceeds, the
two GC rich regions base pair.
This leaves a short poly U
rich region, which cannot pair
strongly enough to hold the
RNA onto the DNA. The
polymerase comes off with it.
3
RNA
polymerase
RNA
Termination
Rho-dependent.
 In vitro, E. coli RNA polymerase holoenzyme
transcribes DNA into a very long RNA.
 The ability of the in vitro reaction to make
natural length RNA is restored by the addition
of a protein factor, called rho ().
RNA transcript length:
By holo polymerase in vitro
In vivo
By holo polymerase + rho
Termination
 Analysis of termination sites dependent on
rho revel a stem loop structure near the 3‘ end
of the RNA, with NO U-rich tale.
 Rho binds to RNA and can, if provided with
ATP, move along the RNA.
 Rho also has ATP-dependent helicase activity.
Termination
Model for rho termination
It has been established that six Rho proteins form a
hexamer to terminate transcription, but the precise
mechanism is not clear.
(1)
The Rho hexamer first binds to the RNA
transcript at an upstream site which is 70-80
nucleotides long and rich in C residues .
Termination
(2)
Upon binding, the Rho hexamer moves along
the RNA in the 5-3 direction, trying to catch
up with the RNA polymerase.
(3)
When the polymerase pauses, which happens
when secondary structures form near the 3 end
of the RNA, rho catches up and melts the
RNA-DNA duplex in the replication bubble,
causing termination.
Termination
DNA
Ribosomes
Transcription and
translation
RNA
Protein
Termination
DNA
Ribosome dissociates
from the RNA when
they encounter a stop
codon.
Termination
DNA
Rho factor binds to
specific sites on
naked RNA.
(i.e. RNA without ribosomes)
Termination
DNA
RNA polymerase
pauses at stem loop,
while rho moves
along RNA, 5-3.
Termination
Rho catches up with polymerase, melting RNADNA duplex, causing polymerase to dissociate.
DNA
Termination
Termination of transcription can serve a role
in regulating gene expression in prokaryotes
This is the subject of the final lecture in this
series.
Suggested reading
Transcription (2000) In: An Introduction to Genetic
Analysis. pp 300-306. Griffiths, A. J. F,. Miller, J.
H., Suzuki, D. T., Lewontin, R. C. and Gelbart, W.
M. (Eds). Freeman and Company, New York.
http://www.nottingham.ac.uk/bennett-lab/lee.html