Fundamental

Molecular Biology
Second Edition

Lisabeth A. Allison

Chapter 10
Transcription in Bacteria

Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Cover photo: Julie Newdoll/www.brushwithscience.com “Dawn1 of the
Double Helix”, oil and mixed media on canvas, © 2003
 Outline
10.1 Introduction
10.2 Mechanism of transcription
10.3 Insights into gene regulation from the lactose (lac) operon
10.4 Mode of action of transcriptional regulators
10.5 Control of gene expression by RNA
10.6 Gene regulatory networks

2
 10.1
• A central event in gene expression is the copying of
the sequence of the template strand of a gene into a
complementary RNA transcript.

• The biochemistry of transcript formation is

straightforward.

• The regulatory mechanisms that have been developed

by bacteria to control transcription are complex and
highly variable.

• RNA polymerase is the enzyme that catalyzes RNA

synthesis.
3
 10.2

• In bacteria, transcription and translation are coupled -

they occur within a single cellular compartment (Fig.
10.1).

• As soon as transcription of the mRNA begins,

ribosomes attach and initiate protein synthesis.

• The whole process occurs within minutes.

• Using DNA as a template, RNA polymerase joins, or

“polymerizes,” nucleoside triphosphates (NTPs) by
phosphodiester bonds from 5' to 3'.
4
5
• Minimal requirements for gene transcription.
– Gene promoter
– RNA polymerase

• Additional factors are required for the

regulation of transcription.

6
 Bacterial promoter structure
• RNA polymerase binds to a region of DNA called a
promoter.

• Bacterial promoters are not absolutely conserved but

they do have a consensus sequence.

• Conserved sequence: When nucleotide sequences of DNA

are aligned with each other, each has exactly the same series of
nucleotides in a given region.

• Consensus sequence: there is some variation in the sequence

but certain nucleotides are present at high frequency.
7
Two distinct consensus sequences

8
Promoter strength
• The relative frequency of transcription initiation.

• Related to the affinity of RNA polymerase for the

promoter region.
• The more closely regions within the promoter
resemble the consensus sequences, the greater the
strength of the promoter.

9
 Structure of bacterial RNA polymerase

• Comprised of a core enzyme plus a transcription

factor called the sigma factor ().
• Together they form the complete, fully functional
enzyme complex called the holoenzyme.

10
 The core enzyme

• The core enzyme catalyzes polymerization.

• 400 kDa & 5 subunits (Fig. 10.3)

• High affinity for most DNA.

• The sequence, structure, and function are

evolutionarily conserved from bacteria to humans.
• X-ray crystallographic studies revealed a crab claw-
like shape.

11
 Sigma factor

• The sigma () factor decreases the nonspecific binding

affinity of the core enzyme.
• Binding results in closing of the core enzyme
“pincers.”
• Primarily involved in recognition of gene promoters.

12
 The sigma factor stimulates tight binding of
RNA polymerase to the promoter

• Shown over 30 years ago using a nitrocellulose

filter binding assay.
• The holoenzyme containing  dissociates more
slowly from template DNA compared with the core
polymerase alone.
• In E. coli the most abundant  factor is 70.

• For expression of some genes, bacterial cells use

alternative  factors.
13
Sigma factor-70

RNA polymerase

14
 Initiation of transcription

Initiation consists of three stages:

1. Formation of a closed promoter complex.

2. Formation of an open promoter complex.

3. Promoter clearance.

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16
Closed promoter complex
• RNA polymerase holoenzyme binds to the promoter
at nucleotide positions 35 and 10.
• The DNA remains double-stranded.

• The complex is reversible.

17
Open promoter complex
• ~18 bp around the transcription start site are melted
to expose the template strand DNA.
• AT rich promoters require less energy to melt.

• Transcription is aided by negative supercoiling of

the promoter region of some genes.
• The open complex is generally irreversible.

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• Transcription is initiated in the presence of NTPs.

• No primer is required for initiation by RNA

polymerase.

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 Promoter clearance
Older “classic” model

  factor release -> new core polymerase

Current model

  factor does not completely dissociate; some

domains are displaced.
• The displaced domains allow the nascent RNA to
emerge from the RNA exit channel.
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21
 Elongation

• After about 9-12 nt of

RNA have been
synthesized, the initiation
complex enters the
elongation stage.

-> conformational change in

the core enzyme

22
Direction of transcription around the E. coli
chromosome

• Of the 50 operons or genes whose transcription

direction is known, 27 are transcribed clockwise
and 23 in the counterclockwise direction around the
circle, using the opposite strand as a template.

• Only one strand of a given operon’s DNA is used as

a template for transcription.

23
• The origin and terminus of replication divide the genome
into oppositely replicated halves or “replichores”

• Most operons or genes are transcribed in the direction of

replication.

• This may lead to fewer collisions of DNA and RNA

polymerase and less topological strain from opposing
supercoils.
• 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.
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• One strand of DNA acts as the template for RNA
synthesis by complementary base pairing.
• The catalytic site of the polymerase has both a
substrate-binding and a product-binding site.
• Transcription always proceeds in the 5′→3′ direction.

• Completion of the single nucleotide addition cycle.

• Shift of the active site of the RNA polymerase by one

position along the template DNA.

25
 Which moves – the RNA polymerase
or the DNA?
Two models

• Model 1: RNA polymerase moves along and the

DNA rotates.
– This is the more widely accepted model.

• Model 2: RNA polymerase remains stationary, and

the DNA moves along and rotates.

26
27
The overall process of transcription has a
significant local effect on DNA structure

• The DNA ahead of the RNA polymerase is wound

more tightly; positive supercoils form.

• Behind the polymerase, DNA becomes less tightly

wound; negative supercoils form.

• Topoisomerase I and gyrase (bacterial

topoisomerase II) resolve this supercoiling and
restore the DNA to its relaxed form.

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 Proofreading
Proofreading by RNA polymerase
– Backtracks 3′→5′
– Pauses
– Nucleolytic cleavage

RNAP: RNA polymerase

29
 Termination of transcription

In most bacteria, there are two types of

terminators:

• Rho-independent

• Rho-dependent

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 Rho-independent termination

• Terminator is characterized by an inverted repeat

consensus sequence.
• Formation of a stem-loop in the exit channel.

• Less stable U-A hybrid helix.

• Polymerase pauses, resulting in transcript release.

31
 Rho-dependent termination
• Terminator is an inverted repeat with no simple
consensus sequence.

• Controlled by the ability of the Rho protein to gain

access to the mRNA.

• Because ribosomes translate mRNA at the same rate

as the mRNA is transcribed, Rho is prevented from
loading onto the newly formed RNA until the end of
a gene or operon. -> ribosome is no longer moving

32
• Rho binds specifically to a C-rich site called a Rho
utilization (rut) site.

• ATP-dependent: polymerase “chasing.”

• Polymerase pauses at the terminator stem-loop

structure, Rho catches up and unwinds the DNA-
RNA hybrid.

• Transcript release.

33
 10.3

The 1959 operon model of Jacob and Monod

• Novel concept of regulatory genes that code for

products that control other genes.

• Model predicted the existence of an unstable RNA

as an intermediate in protein synthesis.

34
 The Jacob-Monod operon model of gene
regulation
• Model arose from experimental observations in
bacteria and phages.

• Study of how phage lambda () can be induced to

switch from lysogenic to lytic state.

• Study of how the enzyme -galactosidase is

produced in bacterial cells only when bacteria need
this enzyme to use the sugar lactose.

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Jacob-Monod operon model

36
• The lac operon provides an example of negative
control of the enzymes involved in lactose
metabolism.

• The lac operon is also regulated by positive control

under certain environmental conditions.

37
 Characterization of the Lac repressor

• Jacob-Monod model also proposed the existence of

a repressor protein.

• Gilbert and Müller-Hill isolated the Lac repressor

and demonstrated that it binds operator DNA (Fig.
10.11).

38
39
 Lactose (lac) operon regulation

• In bacteria, genes are organized into operons.

• An operon is a unit of bacterial gene expression and

regulation, including structural genes and control
elements in DNA recognized by regulatory gene
product(s).

• Transcribed from a single promoter to produce a

single primary transcript of polycistronic mRNA.

40
• In eukaryotes, genes are not typically organized
into operons.

• Exception
– ~15% of genes in Caenorhabditis elegans are
grouped into operons.

– But, each C. elegans pre-mRNA is processed into a

separate mRNA for each gene rather than being
translated as a unit.

41
• Bacteria need to respond swiftly to changes in their
environment, switching from metabolizing one
substrate to another quickly and efficiently.

• Induction is the synthesis of enzymes in response to

the appearance of a specific substrate.

• When provided with a mixture of sugars, bacteria

use glucose first.

42
Unifying theme in gene transcription

• A regulatory protein (trans-acting factor) binds to a

particular sequence of DNA (cis-acting factor)

Trans-acting factor gene Cis-acting DNA

sequence Gene coding region

Transcription

Translation

43
 Lac operon induction
• The lac operon consists of three structural genes, lacZ,
lacY, and lacA.

• lacZ encodes -galactosidase (tetramer), an enzyme

which cleaves lactose into galactose and glucose.

• lacY encodes a lactose permease, part of the transport

system to bring lactose into the cell.

• lacA encodes a transacetylase that rids the cell of toxic

thiogalactosides that get taken up by the permease.

44
• Upstream of the lac operon is the regulatory gene
that codes for the 38-kDa Lac repressor

• Gene name : lower case and italics

– (e.g. baek)
• Protein name: plain type with the first letter in
capitals
– (e.g. Baek)

45
• In the absence of lactose, the Lac repressor binds as
a tetramer to the operator DNA sequence, which
overlaps with the promoter region -> blocks RNA
polymerase from binding to the promoter.

• In the presence of lactose the lac operon is induced.

• Recruitment of RNA polymerase requires formation

of a complex of the cAMP-bound activator protein
CAP, polymerase, and DNA.

46
The Lac repressor binds to the lac operator
CAP: catabolic activator protein – with
cAMP, it recruits RNA polymerase

47
Allolactose – the real inducer

• The real inducer of the lac operon is an alternative

form of lactose called allolactose.

• When -galactosidase cleaves lactose, it rearranges

a small fraction of the lactose to allolactose.

48
Lactose analog: beta-galactosidase
cannot metabolize

49
 Basal transcription of the lac operon

• The basal level of transcription is determined by the

frequency with which RNA polymerase spontaneously
binds the promoter and initiates transcription.

50
51
• The lac operon is transcribed if and only if lactose
is present in the medium.
• But, this signal is almost entirely overridden by the
simultaneous presence of glucose.
• Glucose exerts its effect, in part, by decreasing
synthesis of cAMP which is required for the
activator CAP to bind DNA.
• More importantly, however, glucose inactivates
the lactose permease.
• Without cooperative binding of CAP, RNA
polymerase transcribes the lac genes at low level.
-> basal level 52
 Regulation of the lac operon by Rho

• When cells are starved of amino acids, a Rho-

dependent terminator stops synthesis of mRNA.

53
The lac promoter and lacZ structural gene are
widely used in molecular biology research

• Commonly used reporter gene.

• Expression of heterologous proteins in bacteria.

• In the lab, IPTG is used as an inducer; it interacts

with the Lac repressor, but is not metabolized by -
galactosidase.

54
The lac operon and other operons illustrate
fundamental principles of gene regulation
that are universal.

• Constitutively active RNA polymerase that alone

works with a certain frequency.

• Transcriptional activators increase the frequency of

initiation.

• Transcriptional repressors decrease the frequency of

initiation.
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 10.4
• The repressors and activators are DNA-binding proteins
that undergo allosteric modification.
• Transcriptional repressors and activators are modular
proteins that have domains with distinct functions
(e.g. for DNA binding, ligand binding, and protein-protein
interactions)

• Cooperative binding of proteins to DNA increases their

effective binding constants and allows regulatory proteins
to function at very low concentrations within the cell.
• DNA looping allows multiple proteins to interact with
RNA polymerase, from adjacent and distant sites.

56
Cooperative binding of proteins to DNA

CAP has two major functional domains

– DNA-binding domain
– “Activating” domain which contacts RNA
polymerase

57
• CAP recruits RNA polymerase to the promoter.

• Helps RNA polymerase binds tightly to the

promoter until the polymerase changes from the
closed to open complex.

• One protein might dissociate from the DNA, but

due to its continued interaction with the other
DNA-bound protein, it does not diffuse away and
is more likely to rebind to its DNA site.

58
 Allosteric modification and DNA binding

• Both CAP and the Lac repressor bind to their DNA

sites using a helix-turn-helix (HTH) motif.

• HTH motif: predominant DNA recognition motif

found among E. coli transcriptional regulatory
proteins.

• The recognition helix inserts into the major groove

of the DNA and make sequence-specific contacts
with exposed base pairs.
59
CAP

60
• Allosteric change undergone by CAP upon binding
DNA increases its ability to bind DNA.

• Allosteric change in the Lac repressor upon binding

allolactose decreases its ability to bind DNA.

• The Lac repressor functions as a “dimer of dimers”

• When the Lac repressor is bound to allolactose (or

IPTG) the helix-turn-helix DNA binding motifs become
disordered and dissociate from the binding site.

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62
Interaction of the Lac repressor with lac
operator DNA

• First, binds nonspecifically to DNA.

• The hinge region remains unstructured.
• Then, moves by a “random walk” along the DNA.

63
• Finally, the Lac repressor binds specifically to the
lac operator DNA sequence.
• The hinge region forms an -helix and the DNA
bends by ~36.

Lac repressor

DBD: DNA binding domain of Lac repressor 64

 DNA looping
• DNA looping allows multiple proteins to interact
with RNA polymerase, some from adjacent sites
and some from distant sites.
• The Lac tetramer binds to an upstream auxiliary
operator and the primary operator DNA sequence
forming a DNA loop in between.

• A classic example of DNA looping is found in the

arabinose operon.

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Lac repressor

66
The arabinose operon

• The regulatory protein AraC acts both as a repressor

and activator of transcription.
• Helical-twist experiments were used to confirm DNA
looping of the arabinose operon.
• Addition of half-integral turns to the arabinose operon
sequence interfered with protein-protein interactions
and blocked loop formation.

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 Repressor

Dimeric regulatory protein AraC

Activator

68
 10.5

RNA frequently plays a direct role in

controlling gene expression

• Differential folding of RNA

• Riboswitches

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Differential folding of RNA: transcriptional
attenuation of the tryptophan operon

Regulation of the tryptophan operon occurs by

two mechanisms:
• Transcriptional attenuation
• Conventional protein-mediated repression

70
• Newly synthesized RNA can fold to form either of
two competing hairpin structures:
– antiterminator or terminator

• The leader RNA preceding the antiterminator

contains a 14 nt coding region, trpL, which includes
two tryptophan codons.

71
 Transcriptional
attenuation

Trp

Conventional protein-
mediated repression

72
When bacterial cells have adequate levels of
tryptophan-charged tRNATrp

• The leader peptide (trpL) is synthesized.

• The terminator forms in the leader transcript.

• Transcription is terminated.

73
When cells are deficient in charged tRNA Trp

• The ribosome translating trpL stalls at one of the

tryptophan codons.

• The antiterminator forms and termination is blocked.

• The structural genes involved in tryptophan

biosynthesis are transcribed.

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Conventional protein-mediated repression of
the trp operon

• In the absence of tryptophan, the genes encoding

enzymes for the biosynthesis of tryptophan are
transcribed and translated.

75
When enough tryptophan has been produced:

• Tryptophan binds to the dimeric Trp repressor

protein.

• The Trp repressor binds the trp operator and blocks

access of RNA polymerase to the trp promoter.

76
 Riboswitches
• Specialized domains within certain mRNAs act as
switchable “on-off” elements that selectively bind
metabolites and control gene expression without
the need for protein transcription factors.

• Metabolite sensors and “RNA thermometers”

• Widespread in bacteria.

• In eukaryotes, only one type of riboswitch found

so far in plants and fungi.
77
• Typically found in the 5′ untranslated region (UTR)
of mRNAs.

• Two main structural domains of riboswitches:

– Aptamer: binds to the target metabolite.

– Expression platform: converts metabolite-binding
events into changes in gene expression via
changes in RNA folding. (attenuation)

78
 Metabolite sensors

• Binding of the metabolite usually serves as an

“off” switch, decreasing the expression of the gene
products used to make the metabolite.

• Repression occurs either by terminating

transcription or by preventing translation initiation.

• In some rare cases, the metabolite acts as an “on”

switch.

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Lysine riboswitch (Fig. 10.21A)

• In the absence of lysine, mRNA forms an

antiterminator hairpin and transcription proceeds.

• In the presence of lysine, the aptamer-sensing

domains binds lysine triggering formation of a
transcription terminator.

80
 S-adenosylmethionine
Lysine riboswitch
(SAM) riboswitch

- +
- +

SD: Shine-Dalgano
ribosome-binding sequence

81
Type III S-adenosylmethionine (SAM)
riboswitch (Fig. 10.21B)

• In the absence of SAM, the Shine-Dalgarno

sequence is accessible for ribosome binding and
translation initiation.

• In the presence of SAM, the Shine-Dalgarno

sequence is base-paired in a stem structure,
preventing ribosome binding.

82
 RNA “thermometers”
• Expression of heat shock genes in root nodule
bacteria is regulated by a conserved RNA sequence
element called ROSE (repression of heat-shock gene
expression).

• ROSE is a temperature-sensitive RNA theromometer

riboswitch.
– At low temperature, translation is prevented by an
extended RNA secondary structure.
– At high temperature, the secondary structure partially
melts allowing ribosome access to the mRNA.
83
 Riboswitch ribozymes

• The glmS gene in Bacillus subtilis encodes an enzyme

which generates glucosamine-6-phosphate (GlcN6P)
from fructose-6-phosphate and glutamine.

• glmS mRNA is a highly substrate-specific GlcN6P-

responsive ribozyme.

• Self-destruction of the glmS mRNA inhibits further

production of GlcN6P.

84
(ribozyme)

85
 10.6

• Bacterial regulatory networks have evolved to respond

with remarkable precision to environmental changes.

• Alternative sigma factors coordinate the expression of

different sets of genes or operons.

• Bacteria communicate with each other through the

production of autoinducers.

86
 Alternative sigma factors

• In general, organisms with more varied lifestyles have

more  factors.

• The number of  factors varies from 1 in Mycoplasma

genitalia to more than 63 in Streptococcus coelicolour.

87
• E. coli uses 7 alternative  factors to respond
to some environmental changes: (Table 10.1)
– expression of heat-shock proteins
– expression of flagellar genes

• Bacillus subtillis has 18 different  factors, 5

of which regulate the process of sporulation.

88
89
• In Borrelia burgdorferi, the lyme disease spirochete
carried by ticks, the alternative sigma factor N
stimulates transcription of the rpoS gene encoding the
sigma factor S.

• Activation of N enhances the expression of a large

number of N and S genes that lead to virulence.

90
 Quorum sensing

• Bacteria communicate through the production of

diffusible signal molecules termed autoinducers.

• These molecules are produced at basal levels and

accumulate during growth.

• Once a critical concentration has been reached,

autoinducers can activate or repress a number of
target genes for collective responses.

91
• These responses can include light production, biofilm
formation, or virulence.

• Because the control of gene expression by

autodinducers is cell-density-dependent, this
phenomenon has been called quorum sensing.

92
Autoinducers (3OC6HSL)

93
The LuxIR-type quorum sensing system

• LuxI and LuxR are essential for control of

bioluminescence in Vibrio fischeri.

• These marine bacteria colonize the light organ of the

Hawaiian bobtail squid.

• LuxI catalyzes synthesis of the autoinducer, 3OC6HSL.

94
• When the autoinducer accumulates above threshhold, it
binds the cytoplasmic receptor LuxR.

• When 3OC6HSL is bound to LuxR, the complex binds to

a DNA regulatory sequence (lux box) upstream of the
luciferase operon.

• The luciferase operon is activated.

• The luxAB genes encode the subunits of luciferase, the

enzyme required for light production.

• luxCDE encodes a complex that produces and recycles

the substrate.
95
 Outline
10.1 Introduction
10.2 Mechanism of transcription
10.3 Insights into gene regulation from the lactose (lac) operon
10.4 Mode of action of transcriptional regulators
10.5 Control of gene expression by RNA
10.6 Gene regulatory networks

96