Regulation of gene

expression in Bacteria and

their Viruses

Model of Lac repressor binding to Lac operator

Todays lecture (0223)

Gene regulation
Discovery of the lac system: negative control
(evidence for operator and repressor as an
example)
Catabolite repression of the lac operon: positive
control
Dual positive and negative control: the arabinose
operon
Metabolic pathways and additional levels of
regulation: attenuation

Gene regulation
Bacteria are nutritional opportunists
- acquire compounds they need directly from
environment
or synthesize them using enzymatic
pathways
- preference for not synthesizing due to energy and
cellular resource expenditure
- if need to synthesize nutrients, will only switch on
enzymes when required
Bacteria have regulatory systems that couple gene
expression to sensors that detect compound in
environment

Gene regulation: Basics of

prokaryotic transcriptional
regulation
2 types of protein-DNA interactions required for
transcription regulation:
1) RNA polymerase promoter: determines where
transcription begins
2) Binding of regulatory proteins (activators or
repressors) to specific DNA segments near the
promoter: determines whether promoter-driven
transcription occurs
For some genes, binding of activator to DNA may be
required for transcription to begin gene is under
positive regulation
For others, prevention of binding of repressor to DNA
may be required for transcription of other genes
gene is under negative regulation

Gene regulation: Basics of

prokaryotic transcriptional
regulation
How do activators and repressors regulate
transcription?
- Activators help tether RNA pol to promoter
- Repressors prevent binding of RNA pol. to
promoter or
interfere with its movement along DNA

Gene regulation: Basics of

prokaryotic transcriptional
regulation
How do activators and repressors recognize when
environmental conditions are appropriate for their
actions and act accordingly?

In bacteria repressors bind to operator

sites

Gene regulation: Lac operon

Lac Structural Genes:
-

2 enzymes required for lactose metabolism:

1) Permease (lac Y): transports lactose into cell
2) -galalactosidase (lac Z):

Allolactose (not lactose) =

allosteric effector
(inducer)

3) transacetylase (lac A) is not required for lactose metabolism

Lac Z, Y and A are structural genes that each encode an enzyme
and are transcribed together in a single mRNA molecule
coordinately controlled

Gene regulation: Lac operon

Regulatory components of the lac system:
1) Lac repressor (I): gene encodes a repressor protein
that is diffusible and is located close to the
structural genes. It represses transcription of the lac
structural genes.
2) Lac promoter site (P): region where RNA pol binds to
initiate transcription of the lac structural genes
3) Lac operator site (O): site where Lac repressor binds
and is located between the promoter and the lac Z
gene

Gene regulation: Lac operon

Induction of the lac system: P, O, Z, Y, and A

segments together make up an operon ( =a
segment of DNA encoding a multigenic mRNA as well
as an adjacent promoter and regulatory region).

In the absence of lactose,

the Lac repressor binds to
the operator sequence and
switches off transcription
of the structural genes

Gene regulation: Lac operon

Induction of the lac system: Allolactose or its

analogs, can bind lac repressor and change the shape
of its DNA binding site (allosteric transition) such that
it cannot bind to operator sequences. RNA pol. is
unimpeded in transcribing the structural genes
operon is switched on.

Discovery of the lac system:

Negative control
Jacob and Monods studies on the lac operon were
based on using mutants of the structural and
regulatory genes, and of the regulatory DNA
sequences.
Bacteria is haploid and hence studying the genetic
interactions between mutant and wild type alleles is
hampered.
Made partial bacterial diploids (heterozygotes) by
creating strains transformed with a plasmid (F)
carrying the second copy of the operon

Discovery of the lac system:

Negative control

Synthesis of -Galactosidase and Permease in Haploid

and Heterozygous Diploid Operator Mutants

Oc is a mutant operator site that cannot bind repressor

Discovery of the lac system:

Negative control
Synthesis of -Galactosidase and Permease in Haploid
and Heterozygous Diploid Operator Mutants:
OPERATORS ARE CIS-ACTING (Oc mutation can
only exert its effects on those lac structural
genes on the same DNA segment as the Oc
mutation)

Discovery of the lac system:

Negative control

Synthesis of -Galactosidase and Permease in Haploid

and Heterozygous Diploid Strains Carrying l+ and l

I is a mutant repressor that is inactive

Discovery of the lac system:

Negative control

Synthesis of -Galactosidase and Permease in Haploid

and Heterozygous Diploid Strains Carrying l+ and l:
REPRESSORS ARE TRANS-ACTING (I+ can also
exert its effects on operator sequences that are
on a different DNA segment to the one that has
the I+ allele since it is diffusible)

Catabolite Repression of the lac

Operon: Positive Control
For lac operon to be switched on:
- lactose must be present and glucose must be
absent in the cells environment
Bacteria have preference for utilizing glucose over
any other sugar

if both glucose and lactose are in environment cell

will switch off lac operon

= catabolite repression (glucose is a breakdown

product, or catabolite, of lactose)

Catabolite Repression of the lac

Operon: Positive Control
How does glucose regulate Lac operon?
When glucose levels are high, the levels of cyclic
adenosine monophosphate (cAMP) are low and when
glusoce levels are low cAMP levels are high

Catabolite Repression of the lac

Operon: Positive Control
How does glucose regulate Lac operon?
High cAMP levels can switch on lac operon: cAMP
binds to Catabolite activator protein (CAP) as an
allosteric effector and assists CAP in binding to the
promoter region (at CAP binding site) and activating
transcription by RNA pol.

Catabolite Repression of the lac

Operon: Positive Control
How does glucose regulate Lac operon?
cAMP-CAP may physically interact with RNA pol
and/or when it bends DNA when bound, such binding
of RNA pol to the promoter is facilitated

Summary slide for control of lac

operon (Fig. 11.17)

Read Summary of Lac Operon on

Pgs. 411-412 and look at Figs. 11.17
(negative and positive control of lac
operon: ANNOTATE THIS FIGURE)
and 11.18 (repression and
activation)

Dual Positive and Negative Control:

Arabinose Operon
Arabinose operon
- Structural genes araB, araA and araD encode
enzymes for arabinose breakdown
- araC: activator and repressor (as an activator binds
initiator region araI; as a repressor binds araI and
araO)

Map of Ara operon

Dual Positive and Negative Control:

Arabinose Operon

Binding of both AraCarabinose ad cAMP-CAP

to araI required for RNA
pol to activate
transcription

In the absence of arabinose, AraC

has a different conformation and
binds araI and araO and blocks
binding of RNA pol, repressing
AraC serves as an
activator and as a
transcription
repressor

Metabolic Pathways and additional

levels of regulation: Attenuation
(Trp operon)

Tryptophan (Trp) is a relatively rare amino acid in

proteins

The pathway for synthesis of Trp involves enzymes

encoded by five genes (trpE, trpD, trpC, trpB and
trpA) of the trp operon.

Gene order in the trp operon corresponds to reaction order in the biosynthetic pathway

Metabolic Pathways and additional

levels of regulation: Attenuation
(Trp
operon)
2 mechanisms for regulating transcription of trp operon
1. Intiation of transcription: When Trp levels in a cell are
sufficient, Trp binds Trp repressor (trpR) allowing Trp repressor
to bind operator sequence and switches off transcription.
2. Attenuation (occurs after transcription initiation): Relates to trp
mRNA leader sequence (between transcription start site and
first amino acid of TrpE protein). Remember transcription and
translation are coupled in prokaryotes.
Trp mRNA leader sequence

Determines whether further

transcription will occur once

Metabolic Pathways and additional

levels of regulation: Attenuation
(Trp operon)
Attenuation: the leader sequence has four regions (1-4) that
can base pair forming three stem-loop structures but only two
regions base pair with one another at a given time.
Region 1 contains
trp codons
Region1 Region2
Region 2-Region 3
Region 3-Region 4

Metabolic Pathways and additional

levels of regulation: Attenuation
(Trp operon)
Attenuation responds to levels of charged Trp tRNA
Trp
codons
translated

No charged Trp
tRNA to translate
trp codons

Learning outcomes
Contrast positive and negative gene regulation of
gene expression and explain how both mechanisms
control the activity of the Lac operon.
Identify the trans-acting and cis-acting components of
operons and predict the effect of mutations in these
components on gene expression.
Compare how simple molecules trigger changes in
gene expression of different operons in bacteria.
Explain the mechanism of attenuation of the trp
operon.
Explain how constitutively active mutants of repressor
and operator sequences function with respect to the
lac operon