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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
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 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
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TRANSCRIPTIONAL REGULATION
 The most common way to regulate gene expression in
bacteria is at the transcription initiation level
   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 to regulation by activator proteins
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Allosteric protein
 Regulatory proteins have
two binding sites
  One for a small effector
molecule
The other for DNA
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Activator/Repressor and the effector
molecules
 Small effector molecules affect transcription regulation
  However, these bind to regulatory proteins and 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
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The lac Operon
 An operon is a regulatory unit consisting of a few
structural genes under the control of one promoter
   It encodes polycistronic mRNA that contains the coding
sequence for two or more structural genes
This allows a bacterium to coordinately regulate a group
of genes that encode proteins with a common function
An operon contains several different regions
 Promoter; terminator; structural genes; operator
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  organization and transcriptional regulation of the lac operon
genes
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 β-galactosidase
 Enzymatically cleaves lactose and lactose analogues
 Also converts lactose into allolactose (an isomer)
 lacY  Encodes lactose permease
 Membrane protein required for transport of lactose and analogues
 lacA  Encodes transacetylase
 Covalently modifies lactose and analogues
 Its functional necessity remains unclear
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  Organization and transcriptional regulation of the lac operon
genes
There are two distinct transcriptional units
 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
 There is usually ten tetramer proteins per cell
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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
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RNA pol
cannot access
the promoter
Constitutive
expression
The lac operon is now
repressed
Therefore no allolactose
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The lac operon is now
induced
Some gets converted to allolactose
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Repressor does not completely
inhibit transcription
So very small amounts of the
enzymes are made
The cycle of lac operon induction and repression
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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 sequential use of two sugars by a bacterium is
termed diauxic growth
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The lac Operon Is Also Regulated By an
Activator Protein
  The small effector molecule in catabolite repression
is not glucose
This form of genetic 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)
 Also termed the cyclic AMP receptor protein (CRP)
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The lac Operon Is Also Regulated By an Activator
Protein
 The cAMP-CAP complex is an example of genetic
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
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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
trpR  Encodes the trp repressor protein
 Functions in repression
 trpL  Encodes a short peptide called the Leader peptide
 Functions in attenuation
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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
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Organization of the trp operon and regulation via the trp
repressor protein
   Attenuation occurs in bacteria because of the coupling of
transcription and translation
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
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  Region 2 is complementary to regions 1 and 3
Region 3 is complementary to regions 2 and 4
 Therefore several stem-loops structures are possible
The 3-4 stem loop is
followed by a sequence
of Uracils
It acts as an intrinsic
(ρ-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
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   Therefore, the formation of the 3-4 stem-loop
causes RNA pol to terminate transcription at the
end of the trpL gene
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
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Transcription terminates
just past the trpL gene
Most stable form of
mRNA occurs
Therefore no ribosome associated
with the RNA
Possible stem-loop structures formed from trpL mRNA under
different conditions of translation
Region 1 is blocked
3-4 stem-loop
does not form
RNA pol transcribes
rest of operon
Insufficient amounts
of tRNAtrp
Possible stem-loop structures formed from trpL mRNA under
different conditions of translation
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Sufficient amounts of tRNAtrp
3-4 stem-loop forms
Translation of the trpL mRNA
progresses until stop codon
Ribosome pauses on 2
Region 2 cannot base pair
with any other region
Transcription
terminates
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 (i.e. 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
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TRANSLATIONAL AND POSTTRANSLATIONAL
REGULATION
 Genetic regulation in bacteria is exercised
predominantly at the level of transcription
  However, there are many examples of regulation that occur
at a later stage in gene expression
For example, regulation of gene expression can be
  Translational
Posttranslational
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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
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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 sterically 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
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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
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 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
  Its translation is now blocked
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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
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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 product 3
becomes high
 It will bind to enzyme 1
 Thereby inhibiting its ability to
convert substrate 1 into product 1
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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)
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