CHAPTER 16

Regulation of Gene
Expression in Bacteria and
Bacteriophages
 The lac Operon of E. coli
1. Growth and division genes of bacteria are regulated genes. Their expression is
controlled by the needs of the cell as it responds to its environment with the
goal of increasing in mass and dividing.
2. Genes that generally are continuously expressed are constitutive genes
(housekeeping genes). Examples include protein synthesis and glucose
metabolism.
3. All genes are regulated at some level, so that as resources dwindle the cell can
respond with a different molecular strategy.
4. Prokaryotic genes are often organized into operons that are cotranscribed. A
regulatory protein binds an operator sequence in the DNA adjacent to the gene
array, and controls production of the polycis-tronic (polygenic) mRNA.
5. Gene regulation in bacteria and phage is similar in many ways to the emerging
information about gene regulation in eukaryotes, including humans. Much
remains to be discovered; even in E. coli, one of the most closely studied
organisms on earth, 35% of the genomic ORFs have no attributed function.
 The lac Operon of E. coli
Animation: Regulation of Expression of the lac Operon
Genes
1. An inducible operon responds to an inducer substance
(e.g., lactose). An inducer is a small molecule that joins
with a regulatory protein to control transcription of the
operon.
2. The regulatory event typically occurs at a specific DNA
sequence (controlling site) near the protein-coding
sequence (Figure 16.1).
3. Control of lactose metabolism in E. coli is an example
of an inducible operon.
 Fig. 16.1 General organization of an inducible gene

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 Lactose as a Carbon Source for E. coli
1. E. coli expresses genes for glucose metabolism constitutively, but the genes for
metabolizing other sugars are regulated in a “sugar specific” sort of way. Presence
of the sugar stimulates synthesis of the proteins needed.
2. Lactose is a disaccharide (glucose 1 galactose). If lactose is E. coli’s sole carbon
source, three genes are expressed:
a. β-galactosidase has two functions:
i. Breaking lactose into glucose and galactose. Galactose is converted to glucose, and glucose is
metabolized by constitutively produced enzymes.
ii. Converting lactose to allolactose (an isomerization). Allolactose is involved in regulation of the
lac operon (Figure 16.2).
b. Lactose permease (M protein) is required for transport of lactose across the cytoplasmic
membrane.
c. Transacetylase is poorly understood.
3. The lac operon shows coordinate induction:
a. In glucose medium, E. coli normally has very low levels of the lac gene products.
b. When lactose is the sole carbon source, levels of the three enzymes increase coordinately
(simultaneously) about 1,000-fold.
i. Allolactose is the inducer molecule (Figure 16.2).
ii. The mRNA for the enzymes has a short half-life. When lactose is gone, lac transcription stops,
and enzyme levels drop rapidly.
 Fig. 16.2 Reactions catalyzed by the enzyme -galactosidase

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Experimental Evidence for the
Regulation of lac Genes
1.The experiments of Jacob and Monod
produced an understanding of arrangement and
control of the lac genes.
 Mutations in the Protein-coding
1. Genes
Mutagens produced mutations in the lac structural genes that were used to
map their locations.
a. β-galactosidase is lacZ.
b. Permease is lacY.
c. Transacetylase is lacA.
d. The genes are tightly linked in the order: lacZ-lacY-lacA
2. The type of mutation made a difference in expression of the downstream
genes:
a. Missense mutations affect only the product of the gene with the mutation.
b. Nonsense mutations show polarity (polar mutations), and affect translation of the
downstream genes as well.
3. The interpretation of gene polarity is that ribosomes translate the first gene in
the polycistronic (polygenic) RNA, and finish in proper position to initiate
and translate the next gene. Premature translation termination prevents this
by reducing translation of the downstream genes (Figure 16.3).
 Fig. 16.3 Translation of the polygenic mRNA encoded by lac
utilization genes

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Mutations Affecting the Regulation of
Gene Expression
1. Jacob and Monod found mutants that produced all three lac operon proteins
constitutively, and hypothesized that regulatory mutations had affected the normal
control of gene expression for the operon. Constitutive mutants are in two classes
(Figure 16.4):
a. Mutations in the lac operator (lacO) just upstream from the lacZ gene.
b. Mutations further upstream in the lac repressor gene (lacI).
2. Operator-constitutive (lacOc) mutations were defined by experiments using partial
diploid E. coli F’ strains.
a. An example is the partial diploid F’ lacO+ lacZ- lacY+/lacOc lacZ+ lacY-. (Promoters are
normal for both operons, and the lacA gene is irrelevant to the study.)
b. This strain was tested for β-galactosidase and lactose permease, both in the presence and
absence of the inducer.
c. Without inducer, β-galactosidase is produced, but only inactive permease is made.
i. β-galactosidase is produced from the chromosomal gene under control of the constitutive
promoter.
ii. Permease is produced from the chromosomal gene also, but is inactive because the gene is
mutated.
iii. No products are produced from the F’ DNA, because its promoter is wild-type, and requires
induction for gene expression.
 Fig. 16.4 Organization of the lac genes of E. coli and the associated
regulatory
elements: the lac operon

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 d. With inducer, functional molecules of both β-galactosidase and permease are
produced. This indicates that:
i. β-galactosidase is under constitutive control (on the chromosome).
ii. Permease is under inducible control (on the F’ DNA).
e. In both cases, the promoter controls genes downstream from it on the same DNA
molecule, showing cis-dominance.
3. lacI constitutive genes were also discovered in experiments with partial diploid
strains.
a. An example is the strain lacI+ lacO+ lacZ- lacY+/lacI- lacO+ lacZ+ lacY-, in which
both gene sets have normal operators and promoters.
b. Without inducer, no β-galactosidase or permease is produced.
c. With inducer, both are produced. A lacI+ gene can overcome the lacI- mutation.
d. Since lacI+ and lacI- genes are on different DNA molecules, lacI+ is trans-dominant.
e. Jacob and Monod proposed that lacI+ produces a repressor that controls expression
of both lac operons, making them both inducible.
4. The lac promoter is also affected by mutations (Plac-). Most affect
all three structural genes, which are not made, even when the inducer is present.
a. Plac- mutations affect RNA polymerase binding to the start of the operon.
b. Only genes in the same DNA strand are affected, so Plac- mutations are cis-ominant.
iActivity: Mutations and Lactose Metabolism
 Jacob and Monod’s Operon Model for
the Regulation of lac Genes
1. Jacob and Monod’s model of regulation, with more recent information,
follows:
a. An operon is a cluster of genes that are regulated together. The order of the lac
genes is shown in Figure 16.4, and Figure 16.5 shows the operon when lactose
is absent.
b. The lacI gene has its own constitutive weak promoter and terminator, and
repressor protein is always present in low concentration.
i. The repressor functions as a tetramer (Figure 16.5).
ii. Repressor protein binds the operator (lacO+), and prevents RNA polymerase
initiation to transcribe the operon genes (negative control).
iii. Binding of the repressor to the operator is not absolute, and so an occasional
transcript is made, resulting in low levels of the structural proteins.
c. β-galactosidase in wild-type E. coli growing with lactose as the
sole carbon source converts lactose into allolactose (Figures 16.7 and 16.2).
i. Repressor bound with allolactose bound changes shape (allosteric shift) and
dissociates from the lac operator. Free repressor-allolactose complexes are unable to
bind the operator.
ii. Allolactose induces expression of the lac operon, by removing the repressor and
allowing transcription to occur.
 Fig. 16.5 Functional state of the lac operon in wild-type E. coli
growing in the absence
of lactose

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Fig. 16.6 Molecular model of the lac repressor tetramer

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 16.7 Functional state of the lac operon in wild-type E. coli
growing in the presence of lactose as the sole carbon source
2. The lacOc mutations result in constitutive gene expression.
They are cis-dominant to lacO+, because repressor cannot bind
to the lacOc operator sequence (Figure 16.8).
3. The lacI- mutations change the repressor protein’s
conformation and prevent it from binding the operator,
resulting in constitutive expression of the operon (Figure
16.9).
a. In a partial diploid (lacI+ lacO+ lacZ- lacY+/lacI- lacO+ lacZ+
lacY-), the wild-type repressor (lacI+) is dominant over lacI-
mutants.
b. Defective lacI- repressor can’t bind either operator, but normal
repressor from lacI+ binds both operators and regulates
transcription, resulting in functional β-galactosidase and
permease.
Fig. 16.8a Cis-dominant effect of lacOc mutation in a partial-diploid
strain of E. coli
Fig. 16.8b Cis-dominant effect of lacOc mutation in a partial-diploid
strain of E. coli
Fig. 16.9a Effects of a lacI- mutation
Fig. 16.9b Effects of a lacI- mutation
Fig. 16.9c Effects of a lacI- mutation
4. Additional lacI mutants have been identified:
a. Superrepressor (lacIS) mutants that produce no lac enzymes.
i. The mutant repressor cannot bind allolactose, so lactose does not induce the operon.
ii. The lacIS allele is trans-dominant in partial diploids (lacI+/lacIS) (Figure 16.10). The superrepressor protein binds both
operators and transcription cannot occur.
iii. Normal repressor cannot compete, because superrepressor cannot be induced to fall off.
iv. Low levels of transcription will occur (superrepressor is not covalently bound to the DNA operator sequence), but lacI S
E. coli
cannot use lactose as a carbon source.
b. Dominance (lac-d) mutants have missense mutations at the 5’ end of the lacI gene.
i. In haploid cells, the phenotype is constitutive expression of the lac operon.
ii. In partial diploids, lac-d is trans-dominant. The operon is expressed even in the presence of normal repressor.
iii. Normal repressor functions as a tetramer. Mutant and normal subunits combine randomly.
iv. A tetramer containing one or more mutant subunits cannot bind to operator DNA. Repression does not occur, and so gene
expression is constitutive.
c. Mutations in the lad promoter can increase or decrease the gene¡¦s transcription rate, by altering its affinity for
RNA polymerase. Examples are lacIQ and lacSQ:
i. Both of these mutations raise the transcription rate of the repressor gene. (Q stands for “quantity” and SQ for “super
quantity”)
ii. Large amounts of repressor are produced in these mutants, reducing the efficiency of lac operon induction so that high
levels of lactose are needed.
lii. Overproduction of the lad gene has been useful for isolating and characterizing the repressor molecule.

5. These mutants indicate that repressor has three different recognition interactions:
a. Binding to the operator region.
b. Binding with the inducer (allolactose).
c. Binding of repressor polypeptide subunits to form an active tetramer.
Fig. 16.10 Dominant effect of lacIs mutation over wild-type lacI+
 Positive Control of the lac Operon
Animation: Positive Control of the lac Operon
1. Repressor exerts negative control by preventing transcription. Positive control of
this operon also occurs when lactose is E. coli’s sole carbon source (Figure 16.11).
a. Catabolite activator protein (CAP) binds cyclic AMP (cAMP) (Figure 16.12).
b. CAP-cAMP complex is a positive regulator of the lac operon. It binds the CAP-site, a
DNA sequence upstream of the operon’s promoter.
c. Binding of CAP-cAMP complex causes the DNA to bend, facilitating protein-protein
interactions between CAP and RNA polymerase, and leading to transcription.
2. When both glucose and lactose are in the medium, E. coli preferentially uses
glucose, due to catabolite repression.
a. Glucose metabolism greatly reduces cAMP levels in the cell.
b. The CAP-cAMP level drops, and is insufficient to maintain high transcription of the lac
genes.
c. Even when allolactose has removed the repressor protein from the operator, lac gene
transcription is at very low levels without CAP-cAMP complex bound to the CAP-site.
d. Experimental evidence supports this model. Adding cAMP to cells restored transcription
of the lac operon, even when glucose was present.
Fig. 16.11 Role of cyclic AMP (cAMP) in the functioning of glucose-
sensitive operons such as the lac operon of E. coli
Fig. 16.12 Structure of cyclic AMP (cAMP)
3. The model is that catabolite repression targets adenylate
cyclase (the enzyme that makes cAMP) (Figure 16.12).
a. In E. coli, the phosphorylated form of IIIGlc enzyme activates
adenylate cyclase.
b. Glucose transport into the cell triggers events including
dephosphorylation of IIIGlc.
c. With IIIGlc protein dephosphorylated, adenylate cyclase is
inactivated, and no new cAMP is produced.
4. Catabolic genes for other sugars are also regulated by
catabolite repression. In all cases, a CAP site in their
promoters is bound by a CAP-cAMP complex,
increasing RNA polymerase binding.
 Molecular Details of lac Operon
Regulation
1. The sequences of significant lac regulatory regions are known. DNase
protection by regulatory molecules (e.g., repressor) is useful in these
studies.
2. The lacI DNA sequence shows the expected transcription and translation
signals, except that the start codon is GUG (not AUG). The single base-
pair mutation of IacIQ is also characterized (Figure 16.13).
3. Operon controlling sites (Figure 16.14) were derived from several types of
data:
a. Amino acid sequences of the repressor protein and (β-galactosidase were
known, allowing coding regions for lacI and lacZ+ to be identified.
b. Protection assays identified binding sites for:
i. CAP-cAMP complex.
ii. RNA polymerase.
iii. Repressor protein.
Fig. 16.13 Base pair sequences of the lac operon lacI+ gene promoter
(Plac+) and of the 5' end of the repressor mRNA
4. The lac operon promoter region begins at -84, immediately next to the lacI
gene stop codon, and ends at -8, just upstream from the transcription start
site. Features of the promoter region include:
a. The consensus sequence for CAP-cAMP binding is in two regions: -54 to -58,
and -65 to -69.
b. The RNA polymerase binding site (including a Pribnow box) spans DNA from
-47 to -8, with consensus sequence matches at -10 and -35.
5. The operator is immediately next to the promoter, with repressor protein
protecting DNA from -3 to +21. With repressor bound to the operator,
RNA polymerase can bind but cannot transcribe.
6. The operon transcript begins at +1, which is within the operator region
bound by repressor. (Part of the operator is transcribed.)
a. The β-ga1actosidase gene has a leader region before the start codon.
b. Start codon for 3-galactosidase (AUG) is at + 39 to +41.
c. Several lacOC mutations have been characterized. All are single base pair
substitutions.
7. The lac operon was the first molecular model for gene regulation. Operons
are common in bacteria and phages, but unknown in eukaryotes.
Fig. 16.14 Base pair sequence of the controlling sites, promoter and
operator, for the lactose operon of E. coli
 The trp Operon of E. coli
1.If amino acids are available in the medium, E.
coli will import them rather than make them,
and the genes for amino acid biosynthesis are
repressed. When amino acids are absent, the
genes are expressed and biosynthesis occurs.
2.Unlike the inducible lac operon, the trp operon
is repressible. Generally, anabolic pathways are
repressed when the end product is available.
 Gene Organization of the Tryptophan
Biosynthesis Genes
1. Yanofsky and colleagues characterized the controlling
sites and genes of the trp operon (Figure 16.15).
a. There are 5 structural genes, trpA through E.
b. The promoter and operator are upstream from the trpE gene.
c. Between trpE and the promoter-operator is trpL, the leader
region. Within trpL is the attenuator region (att).
d. The trp operon spans about 7 kb. The operon produces a
polygenic transcript with five structural genes for tryptophan
biosynthesis.
 Fig. 16.15 Organization of controlling sites and the structural genes
of the E. coli trp
operon

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Regulation of the trp Operon
Animation: Attenuation in the trp Operon of E. coli
1. Two mechanisms regulate expression of the trp operon:
a. Repressor/operator interaction.
b. Transcription termination.
2. When tryptophan is present, it will bind to an aporepressor protein (the trpR
gene product).
a. The active repressor (aporepressor plus tryptophan) binds the trp operator, and
prevents transcription initiation.
b. Repression reduces transcription of the trp operon about 70-fold.
3. When tryptophan is limited, transcription is also controlled by
attenuation.
a. Attenuation produces only short (140-bp) transcripts that do not encode structural
proteins.
b. Termination occurs at the attenuator site within the trpL region.
c. The proportion of attenuated transcripts to full-length ones is related to tryptophan
levels, with more attenuated transcripts as the tryptophan concentration increases.
d. Attenuation can reduce trp operon transcription 8- to 10-fold. Together, repression
and attenuation regulate trp gene expression over a 560- to 700-fold range.
4. The molecular model for attenuation:
a. Translation of the trpL gene produces a short polypeptide.
Near the stop codon are two tryptophan codons.
b. Within the leader mRNA are four regions that can form
secondary structures by complementary base-pairing
(Figure 16.16).
i. Pairing of sequences 1 and 2 creates a transcription pause signal.
ii. Pairing of sequences 3 and 4 is a transcription termination signal.
iii. Pairing of 2 and 3 is an antitermination signal, and so
transcription will continue.
c. Tight coupling of transcription and translation in
prokaryotes makes control by attenuation possible.
i. RNA polymerase pauses when regions 1 and 2 base pair just after
they are synthesized (Figure 16.16).
 ii. During the pause, a ribosome loads onto the mRNA and begins translation of
the leader peptide. Ribosome position is key to attenuation:
(1) When tryptophan (Trp) is scarce:
(a) Trp-tRNAs are unavailable, and the ribosome stalls at the Trp codons in the
leader sequence, covering attenuator region 1.
(b) When the ribosome is stalled in attenuator region 1, it cannot base pair with
region 2. Instead, region 2 pairs with region 3 when it is synthesized.
(c) If region 3 is paired with region 2, it is unable to pair with region 4 when it is
synthesized. Without the region 3-4 terminator, transcription continues
through the structural genes.
(2) When Trp is abundant:
(a) The ribosome continues translating the leader peptide, ending in region 2.
This prevents region 2 from pairing with region 3, leaving 3 available to pair
with region 4.
(b) Pairing of regions 3 and 4 creates a rho-independent terminator known as the
attenuator. Transcription ends before the structural genes are reached.

5. Genetic evidence for attenuation includes:

a. Mutations in the leader sequence within regions 3 or 4 disrupt base
pairing and decrease the efficiency of termination (Figure 16.18)
b. Changes in the Trp codons of the leader peptide sequence, so that
they encode a different amino acid, cause attenuation controlled by
levels of the amino acid specified by the mutant codon, not by Trp
 Fig. 16.16 Four regions of the trp operon leader mRNA and the
alternate secondary
structures they can form by complementary base-pairing

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Fig. 16.17a Models for attenuation in the trp operon of E.coli

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Fig. 16.17b Models for attenuation in the trp operon of E.coli

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Fig. 16.18 In the trpL region, mutation sites that show less efficient
transcription at
the attenuator site

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Regulation of Other Amino Acid
Biosynthesis Operons

1.Attenuation is involved in regulating many

operons.
a. When the operon is for amino acid biosynthesis,
the leader sequence always includes codons for
that amino acid.
b.Other operons regulated by attenuation include
rRNA (rrn) and E. coli ampicillin resistance
(ampC).
 Fig. 16.19 Predicted amino acid sequences of the leader peptides of a
number of
attenuator-controlled bacterial operons

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Regulation of Gene Expression in
Phage Lambda
1. Phage use many bacterial components for replication,
and control their use with phage gene products.
2. Bacteriophage λ has two possible pathways when it
enters its E. coli host:
a. The lytic cycle, in which the phage takes over the cell and
produces progeny phage.
b. The lysogenic cycle, where phage chromosome is inserted
into the E. coli chromosome, and replicates with the
bacterial genome.
 Fig. 16.20 A map of phage , showing the major genes

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 Early Transcription Events
1. The λ chromosome is linear, with “sticky” ends used to
circularize it in the host cell. The regulatory system for
choosing between the lytic and lysogenic pathways is
contained in the λ chromosome.
a. First, transcription begins at promoters PL (leftward
transcription) and PR (rightward) (Figure 16.21).
i. The first gene transcribed from PR is cro (control of repressor and other).
The Cro protein is involved in the genetic switch to the lytic pathway.
ii. The first protein transcribed from PL is N, which is a transcription
antiterminator that allows RNA synthesis to go through termination
regions into the early genes.
(1) N protein allows expression of the cII protein, which in turn activates:
(a)cI (λ repressor)
(b) O and P (DNA replication proteins).
(c)Q (activation of late genes for lysis and phage particle proteins, only
when Q protein accumulates to certain levels).
 Fig. 16.21 Expression of  genes after infecting E. coli and the
transcriptional events
that occur when either the lysogenic or lytic pathways are
followed

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Fig. 16.21 Expression of  genes after infecting E. coli and the
transcriptional events
that occur when either the lysogenic or lytic pathways are
followed (cont.)

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 The Lysogenic Pathway
1. Early transcription events determine whether the lytic or lysogenic pathway
occurs.
2. The lysogenic pathway results when cII and cIII are expressed.
a. The action of cII and cIII proteins activates the PRE promoter, causing transcription
of the cI (λ repressor) gene.
b. λ repressor binds to 2 operator regions, OL and OR, whose sequences overlap PL
and PR, respectively. This prevents transcription from PL and PR.
c. Transcription of N and cro genes is blocked, and concentrations of their proteins
drop rapidly.
d. In addition, repressor bound to OR causes more repressor mRNA to be made from
another promoter, PRM.
e. High levels of repressor cause lysogeny by binding operators OL and OR.
f. Integrase protein is used to integrate λ DNA into the E. coli chromosome. Integrase
transcript is initiated at the PI promoter, which is controlled by the cII protein.
g. As cII concentration drops, the PI promoter is shut down, leaving PRM as the only
active promoter.
3. Thus, the lysogenic pathway occurs when enough λ repressor is
made to turn off early promoters. Lytic genes, including Q, are not expressed.
Without the Q protein, phage coat and lysis proteins are not produced.
 The Lytic Pathway
1. An example of induction of the lytic pathway is exposure to UV
light.
a. UV causes the bacterial RecA protein (normally used in DNA repair) to
stimulate the λ repressor proteins to autocleave and become inactivated.
b. Absence of repressor at OR allows transcription of the cro gene.
c. Cro protein decreases RNA synthesis from PL and PR, reducing
synthesis of cII, therefore blocking synthesis of λ repressor.
d. Transcription from PR is also decreased, but Q protein levels are
sufficient to allow transcription of the late genes needed for the lytic
pathway.
2. Thus, λ uses complex regulatory systems to control entry into the
lytic or lysogenic pathway. The decision depends on competition
between the repressor and the Cro protein.
a. If repressor dominates, lysogeny takes place.
b. If the Cro protein dominates, the lytic pathway occurs.