Lecture PowerPoint to accompany

Molecular Biology
Fourth Edition

Robert F. Weaver

Chapter 6
The Mechanism of
Transcription in
Bacteria
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 Key points
• Transcription is the process in which a gene's DNA sequence is
copied (transcribed) to make an RNA molecule.
• RNA polymerase is the main transcription enzyme.
• Transcription begins when RNA polymerase binds to
a promoter sequence near the beginning of a gene (directly or
through helper proteins).
• RNA polymerase uses one of the DNA strands (the template
strand) as a template to make a new, complementary RNA
molecule.
• Transcription occurs in three phases known as initiation,
elongation and termination.
• Termination depends on sequences in the RNA, which signal
that the transcript is finished. 6-2
6-3
 6.1 RNA Polymerase Structure
By 1969 SDS-PAGE of RNA polymerase from E.
coli had shown several subunits
– 2 very large subunits are b (150 kD) and b’ (160
kD)
– Sigma (s) at 70 kD
– Alpha (a) at 40 kD – 2 copies present in
holoenzyme
– Omega (w) at 10 kD
• Was not clearly visible in SDS-PAGE, but seen in
other experiments
• Not required for cell viability or in vivo enzyme activity
• Appears to play a role in enzyme assembly
6-4
 Sigma as a Specificity Factor
• Core enzyme without the s subunit could not
transcribe viral DNA, yet had no problems with
highly nicked calf thymus DNA
• With s-subunit, the holoenzyme worked equally
well on both types of DNA

6-5
 Testing Transcription
• Core enzyme transcribes both DNA strands
• Without s-subunit the core enzyme has basic
transcribing ability but lacks specificity

6-6
 6.2 Promoters
• Nicks and gaps are good sites for RNA
polymerase to bind nonspecifically
• Presence of the s-subunit permitted
recognition of authentic RNA polymerase
binding sites
• Polymerase binding sites are called
promoters
• Transcription that begins at promoters is
specific, directed by the s-subunit
6-7
 RNA Polymerase Binding
Hinkle and Chamberlin proposed:
• RNA polymerase holoenzyme binds DNA loosely
at first
– Binds at promoter initially
– Scans along the DNA until it finds one
• Complex with holoenzyme loosely bound at the
promoter is a closed promoter complex as DNA
is in a closed ds form
• Holoenzyme can then melt a short DNA region
at the promoter to form an open promoter
complex with polymerase bound tightly to DNA
6-8
 Polymerase/Promoter Binding
• Holoenzyme binds DNA
loosely at first
• Complex loosely bound at
promoter = closed
promoter complex,
dsDNA in closed form
• Holoenzyme melts DNA
at promoter forming open
promoter complex -
polymerase tightly bound

6-9
6-10
 Core Promoter Elements
• There is a region common to bacterial promoters
described as 6-7 bp centered about 10 bp upstream of
the start of transcription = -10 box
• Another short sequence centered 35 bp upstream is
known as the -35 box
• Comparison of thousands of promoters has produced a
consensus sequence for each of these boxes

6-11
 Promoter Strength
• Consensus sequences:
– -10 box sequence approximates TATAAT
– -35 box sequence approximates TTGACA
• Mutations that weaken promoter binding:
– Down mutations
– Increase deviation from the consensus
sequence
• Mutations that strengthen promoter binding:
– Up mutations
– Decrease deviation from the consensus
sequence
6-12
 6.3 Transcription Initiation
• Transcription initiation was assumed to
end as RNA polymerase formed 1st
phosphodiester bond
• Carpousis and Gralla found that very small
oligonucleotides (2-6 nt long) are made
without RNA polymerase leaving the DNA
• Abortive transcripts such as these have
been found up to 10 nt

6-13
Stages of Transcription Initiation
• Formation of a closed
promoter complex
• Conversion of the closed
promoter complex to an
open promoter complex
• Polymerizing the early
nucleotides – polymerase
at the promoter
• Promoter clearance –
transcript becomes long
enough to form a stable
hybrid with template
6-14
 The Functions of s
• Gene selection for transcription by s
causes tight binding between RNA
polymerase and promoters
• Tight binding depends on local melting of
DNA that permits open promoter complex
• Dissociation of s from core after
sponsoring polymerase-promoter binding

6-15
 Sigma Stimulates Transcription
Initiation
• Stimulation by s appears
to cause both initiation
and elongation
• Or stimulating initiation
provides more initiated
chains for core
polymerase to elongate

6-16
 Reuse of s

• During initiation s can be recycled for additional

use in a process called the s cycle
• Core enzyme can release s which then
associates with another core enzyme
6-17
Sigma May Not Dissociate from
Core During Elongation
• The s -factor changes its relationship to
the core polymerase during elongation
• It may not dissociate from the core
• May actually shift position and become
more loosely bound to core

6-18
 Region of Early Promoter
Melted by RNA Polymerase

6-19
 6.4 Elongation
• After transcription initiation is
accomplished, core polymerase
continues to elongate the RNA
• Nucleotides are added sequentially, one
after another in the process of elongation

6-20
Function of the Core Polymerase
• Core polymerase contains the RNA
synthesizing machinery
• Phosphodiester bond formation involves
the b- and b’-subunits
• These subunits also participate in DNA
binding
• Assembly of the core polymerase is a
major role of the a-subunit
6-21
 Topology of Elongation
• Elongation of transcription involves
polymerization of nucleotides as the RNA
polymerase travels along the template DNA
• Polymerase maintains a short melted region of
template DNA
• DNA must unwind ahead of the advancing
polymerase and close up behind it
• Strain introduced into the template DNA is
relaxed by topoisomerases

6-22
6.5 Termination of Transcription
• When the polymerase reaches a
terminator at the end of a gene it falls off
the template and releases the RNA
• There are 2 main types of terminators
– Intrinsic terminators function with the RNA
polymerase by itself without help from other
proteins
– Other type depends on auxiliary factor called
r, these are r-dependent terminators
6-23
 Rho-Independent Termination
• Intrinsic or r-independent termination
depends on terminators of 2 elements:
– Inverted repeat followed immediately by
– T-rich region in nontemplate strand of the
gene
• An inverted repeat predisposes a
transcript to form a hairpin structure

6-24
 Inverted Repeats and Hairpins
• The repeat at right is
symmetrical around
its center shown with
a dot
• A transcript of this
sequence is self-
complementary
– Bases can pair up to
form a hairpin as seen
in the lower panel

6-25
 Model of Intrinsic Termination
Bacterial terminators act by:
• Base-pairing of
something to the
transcript to destabilize
RNA-DNA hybrid
– Causes hairpin to form
• Causing the transcription
to pause
– Causes a string of U’s to
be incorporated just
downstream of hairpin 6-26
 Rho-Dependent Termination
• Rho caused depression of the ability of
RNA polymerase to transcribe phage
DNAs in vitro
• This depression was due to termination of
transcription
• After termination, polymerase must
reinitiate to begin transcribing again

6-27
 Rho Affects Chain Elongation
• There is little effect of r on transcription
initiation, if anything it is increased
• The effect of r on total RNA synthesis is a
significant decrease
• This is consistent with action of r to
terminate transcription forcing time-
consuming reinitiation

6-28
 Rho Causes Production of
Shorter Transcripts
• Synthesis of much smaller RNAs occurs in
the presence of r compared to those
made in the absence
• To ensure that this due to r, not to RNase
activity of r, RNA was transcribed without
r and then incubated in the presence of r
• There was no loss of transcript size, so no
RNase activity in r
6-29
 Rho Releases Transcripts from
the DNA Template
• Compare the sedimentation of transcripts
made in presence and absence of r
– Without r, transcripts cosedimented with the
DNA template – they hadn’t been released
– With r present in the incubation, transcripts
sedimented more slowly – they were not
associated with the DNA template
• It appears that r serves to release the
RNA transcripts from the DNA template
6-30
 Mechanism of Rho
• No string of T’s in the r-
dependent terminator,
just inverted repeat to
hairpin
• Binding to the growing
transcript, r follows the
RNA polymerase
• It catches the
polymerase as it pauses
at the hairpin
• Releases transcript from
the DNA-polymerase
complex by unwinding
the RNA-DNA hybrid 6-31
Transcription in Prokaryotes

6-32