MICRIBIAL GENETICS

MOLECULAR MECHANISM OF
GENE REGULATION
INTRODUCTION
 The term gene regulation means that the level of
gene expression can vary under different conditions

 Genes that are unregulated are termed constitutive

 They have essentially constant levels of expression
 Frequently, constitutive genes encode proteins that are
continuously necessary for the survival of the organism

 The benefit of regulating genes is that encoded

proteins will be produced only when required
INTRODUCTION
 Gene regulation is important for cellular processes
such as
 1. Metabolism
 2. Response to environmental stress
 3. Cell division

 Regulation can occur at any of the points on the

pathway to gene expression
OVERVIEW OF TRANSCRIPTIONAL
REGULATION
 The most common way to regulate gene expression in
bacteria is by influencing the initiation of transcription
 The rate of RNA synthesis can be increased or decreased

 Transcriptional regulation involves the actions of two main

types of regulatory proteins
 Repressors  Bind to DNA and inhibit transcription

 Activators  Bind to DNA and increase transcription

 Negative control refers to transcriptional regulation by

repressor proteins
 Positive control refers to regulation by activator proteins
 Small effector molecules affect transcription regulation
 These bind to regulatory proteins but not to DNA directly

 In some cases, the presence of a small effector molecule

may increase transcription
 These molecules are termed inducers
 They function in two ways
 Bind activators and cause them to bind to DNA
 Bind repressors and prevent them from binding to DNA
 Genes that are regulated in this manner are termed inducible

 In other cases, the presence of a small effector molecule

may inhibit transcription
 Corepressors bind to repressors and cause them to bind to DNA
 Inhibitors bind to activators and prevent them from binding to DNA
 Genes that are regulated in this manner are termed repressible
 Regulatory proteins have two binding sites
 One for a small effector molecule
 The other for DNA
REGULATION OF THE lac OPERON
 At the turn of the 20th century, scientists made the
following observation
 A particular enzyme appears in the cell only after the cell
has been exposed to the enzyme’s substrate
 This observation became known as enzyme adaptation

 François Jacob and Jacques Monod at the Pasteur

Institute in Paris were interested in this phenomenon
 They focused their attention on lactose metabolism in
E. coli
 The lac Operon
 An operon is a regulatory unit consisting of a few
structural genes under the control of one promoter
 An operon encodes a polycistronic mRNA that contains the
coding sequence for two or more structural genes
Cistron: the smallest unit of genetic material
which coded for a single polypeptide; Based on
Cis-trans test
 This allows a bacterium to coordinately regulate a group of
genes that encode proteins with a common functional goal

 An operon contains several important DNA sequences

 Promoter; terminator; structural genes; operator
 lac operon
 There are two distinct transcriptional units
 1. The actual lac operon

 a. DNA elements
 Promoter  Binds RNA polymerase
 Operator  Binds the lac repressor protein
 CAP site  Binds the Catabolite Activator Protein (CAP)
 b. Structural genes
 lacZ  Encodes b-galactosidase
 Enzymatically cleaves lactose and lactose analogues

 Also converts lactose to allolactose (an isomer)

 lacY  Encodes lactose permease

 Membrane protein required for transport of lactose and analogues

 lacA  Encodes galactoside transacetylase

 Covalently modifies lactose and analogues

 Its functional necessity remains unclear

 2. The lacI gene
 Not considered part of the lac operon

 Has its own promoter, the i promoter

 Constitutively expressed at fairly low levels

 Encodes the lac repressor

 The lac repressor protein functions as a tetramer

 Only a small amount of protein is needed to repress the lac operon

The lac Operon Is Regulated By
a Repressor Protein
 The lac operon can be transcriptionally regulated
 1. By a repressor protein
 2. By an activator protein

 The first method is an inducible, negative control

mechanism
 It involves the lac repressor protein
 The inducer is allolactose
 It binds to the lac repressor and inactivates it
 RNA pol can’t
transcribe
lacZ, lacY, and
lacA

Constitutive
expression

The lac operon is

repressed

Therefore no allolactose
The lac operon is now
induced

Some gets converted to allolactose

The lac Operon Is Also Regulated
By an Activator Protein
 The lac operon can be transcriptionally regulated in
a second way, known as catabolite repression
 When exposed to both lactose and glucose
 E. coli uses glucose first, and catabolite repression
prevents the use of lactose
 When glucose is depleted, catabolite repression is
alleviated, and the lac operon is expressed
The lac Operon Is Also Regulated
By an Activator Protein
 The small effector molecule in catabolite repression
is not glucose

 This form of regulation involves a small molecule,

cyclic AMP (cAMP)
 It is produced from ATP via the enzyme adenylyl cyclase

 cAMP binds an activator protein known as the Catabolite

Activator Protein (CAP)
The lac Operon Is Also Regulated
By an Activator Protein
 The cAMP-CAP complex is an example of
transcriptional regulation that is inducible and under
positive control
 The cAMP-CAP complex binds to the CAP site near the
lac promoter and increases transcription

 In the presence of glucose, the enzyme adenylyl

cyclase is inhibited

This decreases the levels of cAMP in the cell
 Therefore, cAMP is no longer available to bind CAP
 Transcription rate decreases
The lac Operon Has Three
Operator Sites for the lac Repressor
 Detailed genetic and crystallographic studies have
shown that the binding of the lac repressor is more
complex than originally thought

 In all, three operator sites have been discovered

 O1  Next to the promoter (slightly downstream)
 O2  Downstream in the lacZ coding region
 O3  Slightly upstream of the promoter
 Repression is 1,300 fold
Therefore, transcription is 1/1,300
the level when lactose is present

No repression
Constitutive expression
The identification of three lac operator sites
 The results supported the hypothesis that the lac
repressor must bind to two of the three operators to
cause repression
 It can bind to O1 and O2 , or to O1 and O3
 But not O2 and O3
 If either O2 or O3 is missing, maximal repression is not
achieved

 Binding of the lac repressor to two operator sites

requires that the DNA form a loop
 A loop in the DNA brings the operator sites closer together
 This facilitates the binding of the repressor protein
 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

This loop inhibits RNA

polymerase movement past O1

o3 o1

Each repressor lac repressor tetramer Each repressor

dimer binds to one dimer binds to one
operator site operator site

o1 o2

(a) DNA loops caused by

the binding of the lac repressor
REGULATION OF THE trp OPERON
 The trp operon (pronounced “trip”) is involved in the
biosynthesis of the amino acid tryptophan
 The genes trpE, trpD, trpC, trpB and trpA encode
enzymes involved in tryptophan biosynthesis

 The genes trpR and trpL are involved in regulation of the

trp operon
 trpR  Encodes the trp repressor protein
 Functions in repression
 trpL  Encodes a short peptide called the Leader peptide
 Functions in attenuation
 RNA pol can bind
to the promoter

Cannot bind to
the operator site

Organization of the trp operon and regulation via the trp

repressor protein
Organization of the trp operon and regulation via the trp
repressor protein
 Another mechanism
of regulation

Organization of the trp operon and regulation via the trp

repressor protein
 Attenuation can occur in bacteria because transcription and
translation are coupled

 During attenuation, transcription actually begins but it is

terminated before the entire mRNA is made
 A segment of DNA, termed the attenuator, is important in facilitating
this termination
 In the case of the trp operon, transcription terminates shortly past
the trpL region
 Thus attenuation inhibits the further production of tryptophan

 The segment of trp operon immediately downstream from

the operator site plays a critical role in attenuation
 The first gene in the trp operon is trpL
 It encodes a short peptide termed the Leader peptide
 Region 2 is complementary to regions 1 and 3
 Region 3 is complementary to regions 2 and 4
 Therefore multiple stem-loops structures are possible

The 3-4 stem loop is

followed by a sequence
of Uracils

It acts as an intrinsic
(r-independent) terminator

These two codons provide a way

to sense if there is sufficient
tryptophan for translation

Sequence of the trpL mRNA produced during attenuation

 The formation of the 3-4 stem-loop causes RNA pol
to pause, allowing the U-rich sequence to
dissociate from the DNA

 Conditions that favor the formation of the 3-4

stem-loop rely on the translation of the trpL mRNA

 There are three possible scenarios

 1. No translation
 2. Low levels of tryptophan
 3. High levels of tryptophan
 Transcription
terminates just
past the trpL
gene

Most stable form of

mRNA occurs

Therefore no ribosome are

associated with the RNA

Possible stem-loop structures formed from trpL mRNA under

different conditions of translation
 Region 1 is blocked

RNA pol transcribes

rest of operon

3-4 stem-loop
does not form

Insufficient amounts
of tRNAtrp

Figure 14.13 Possible stem-loop structures formed from trpL mRNA under
different conditions of translation
14-47
 Sufficient amounts of tRNAtrp

Translation of the trpL mRNA

progresses until stop codon
Ribosome pauses on region 2 3-4 stem-loop forms
Region 2 cannot base pair
with any other region

Transcription
terminates
Attenuation is found
in several other
operons involved in
amino acid synthesis

Possible stem-loop structures formed from trpL mRNA under

different conditions of translation
Inducible vs Repressible Regulation
 The study of many operons revealed a general trend
concerning inducible versus repressible regulation

 Operons involved in catabolism (ie. breakdown of a

substance) are typically inducible
 The substance to be broken down (or a related compound) acts
as the inducer

 Operons involved in anabolism (ie. biosynthesis of a

substance) are typically repressible
 The inhibitor or corepressor is the small molecule that is the
product of the operon
 TRANSLATIONAL AND
POSTTRANSLATIONAL REGULATION
 Genetic regulation in bacteria is exercised
predominantly at the level of transcription
 However, there are many examples of regulation that
occurs at a later stage in gene expression

 For example, regulation of gene expression can be

 Translational
 Posttranslational
Translational Regulation
 For some bacterial genes, the translation of mRNA
is regulated by the binding of proteins

 A translational regulatory protein recognizes

sequences within the mRNA

 In most cases, these proteins act to inhibit

translation
 These are known as translational repressors
Translational Regulation
 Translational repressors inhibit translation in one of
two ways
 1. Binding next to the Shine-Dalgarno sequence and/or
the start codon
 This will sterictlly hinder the ribosome from initiating translation

 2. Binding outside the Shine-Dalgarno/start codon region

 They stabilize an mRNA secondary structure that prevents initiation

 Translational repression is also found in eukaryotes

Translational Regulation
 A second way to regulate translation is via the
synthesis of antisense RNA
 An RNA strand that is complementary to mRNA

 Consider, for example, the trait of osmoregulation

 The ability to control the amount of water inside the cell
 Must be regulated to prevent shrinkage or lysis

 The protein ompF in E. coli is important in osmoregulation

 This outer membrane protein is encoded by the ompF gene
 OmpF protein is preferentially produced at low osmolarity
 At high osmolarity its synthesis is decreased
 The expression of another gene, termed micF, is responsible
for inhibiting the ompF gene at high osmolarity
 micF RNA does not code for a protein
 It is, however, complementary to ompF mRNA
 It is thus termed antisense RNA

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Stem-loop structure

Antisense RNA
from micF gene
3′ 5′

ompF mRNA
5′ 3′

Its translation is now blocked

Posttranslational Regulation
 A common mechanism to regulate the activity of
metabolic enzymes is feedback inhibition

 The final product in a pathway often can inhibit the

an enzyme that acts early in the pathway
 Enzyme 1 is an allosteric
enzyme, which means it
contains two different binding
sites
 Catalytic site  binds
substrate
 Regulatory site  binds final
product of the pathway

 If the concentration of the

final product becomes high
 It will bind to enzyme 1
 Thereby inhibiting its ability to
convert substrate 1 into
intermediate 1
Posttranslational Regulation
 A second strategy to control the function of proteins
is by the covalent modification of their structure
 Some modifications are irreversible
 Proteolytic processing
 Attachment of prosthetic groups, sugars, or lipids
 Other modifications are reversible and transiently
affect protein function
 Phosphorylation (–PO4)
 Acetylation (–COCH3)
 Methylation (–CH3)