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BLGY1232 Control of bacterial gene expression
Why study it?
 Many fundamental issues concerning the nature of the gene and gene expression
were originally elucidated in bacteria and their phages
 Easy to grow rapidly and in large quantities - facilitates biochemical approaches
 Machinery for carrying out DNA replication, gene transcription, protein synthesis is
simpler (fewer components) in bacteria than higher cells, though common principles
are conserved
 Bacteria are important bio-factories and sources of therapeutics
 Test-bed for systems biology; fundamental interest
Regulation of gene expression
 Transcription, translation and mRNA degradation are coupled
 Levels of specific protein can be controlled by;
 Regulation of mRNA synthesis; rate of transcription initiation and frequency
of transcriptional read-through
 Regulation of mRNA degradation
 Regulation of protein synthesis; rate of translation initiation
 Regulation of protein degradation
Gene transcription: the fundamental issues
 Genes may be expressed throughout the life of the cell and this is termed by
constitutive gene expression – different genes can be expressed constitutively to
different levels
 Bacteria – changes in transcription of specific genes in response to availability of
specific nutrients and to stress and some to development
 A bacterial gene is only expressed when there is a specific demand for its product
Transcription

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Involves melting of the DNA template
RNA polymerase synthesizes RNA strands using DNA as template
DNA melts open at site of transcription - the “transcription bubble”
The newly synthesized RNA chain is extended in the 5’ to 3’ direction
The strand of DNA that is complementary to the newly synthesized RNA chain is
called the “template strand” (because it is used as the template in the RNA synthesis
mechanism)
Other names for the template strand include non-coding or antisense strand
RNA sequence is complementary to the template strand and identical to the coding
strand

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The elements that regulate the transcription and translation of a gene are described
by the sequence on the coding strand

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The DNA nucleotide encoding the beginning of the RNA chain is called the
transcription start site) and is designated the “+1” position
...
e
...
Downstream sequences
are allotted positive values
...
When referring to a specific position in the upstream sequence, this is
given a negative value
...

Single point mutations in these small sequences can affect the frequency of
transcription initiation
Sigma factor determines promotor specificity (70 binds to -35 and -10 boxes of
‘vegetative’ E
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e
...
coli there are at least 6 known alternatives the
alternative sigma factors of E
...
Only a subset of the phage genes are transcribed using E
...
One of these so called ‘early’ genes encodes an inhibitor (anti-sigma factor)
of 70 (thereby shuts off expression of host genes), while another encodes a
new  factor
...
This  factor, 33 directs transcription of a ‘middle’ set of T4 genes, which
are primarily concerned with replication of the viral genome
...
The middle genes include one that encodes yet another  factor
...
This  factor, 55, displaces 33 (thus stops replication) and directs
transcription of the ‘late’ T4 genes, which encode the structural proteins of
the virus
...
subtilis – major player Richard
Losick (Harvard)
Another example of a virulent phage that encodes an alternative sigma factor is
SPO1, which infects B
...
coli, two classes of terminator sequences, which respectively do or do not
require an additional protein subunit, the r (rho) subunit
...
This pausing of
the polymerase coincides with transcription of the oligo (U) sequence
...

 The r-dependent terminators do form strong hairpins, but these are not as strong,
and do not have the run of U’s  Rho appears to recognize a site upstream of point
of termination and then chase RNAP (5’ to 3’ direction); and then using a helicase
activity, cause dissociation of RNA in the transcription bubble
Translation
 Translation is mediated by ribosomes: a complex composed of RNA molecules and
many polypeptide chains
...

 The subunits contain the 16S and 23S rRNA molecules
...

 The ribosome binding site has a degree of complementarity to a segment of 16S
rRNA
...

 Similarity to consensus can determine efficiency of translation
 Translation initiation is a major point of gene regulation in eubacteria
 Segments encoding RBS can be recognised in the DNA
...

During translation, ribosome moves along an mRNA molecule in the 5’ to 3’
direction
...

the codon is recognised by an aminoacyl transfer RNA – tRNA – bearing the
appropriate amino acid

The genetic code

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Codon usage is universal with only a few exceptions

Translational termination
 Nascent polypeptide chain stays attached to the ribosome until translation
terminates which happens when the triplet is one of the three (UAG, UAA and UGA)
stop codons or nonsense codons
...

 Stop codons are not recognised by a special tRNA, but polypeptides called release
factors
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coli is grown on a mixture of glucose and lactose, it utilises the glucose first
...

An early model, called the instruction hypothesis, stated that all proteins were
present in a cell, but in the absence of an inducer were not properly folded and were
inactive
...

 The core of their model was that a response was mediated by regulating the
level of specific cellular proteins
...

The utilization of lactose;
 The specific activity (enzyme units per mg of total protein) of enzymes
required for glucose metabolism (e
...
via glycolysis) does not alter
significantly depending on the growth conditions
...
g
...

 The specific activity of -galactosidase, which breaks lactose into glucose +
galactose, is ~1000-fold higher when cells are grown in lactose compared
with glucose
 In the absence of lactose there are only 1-5 molecules of each of the Lac
proteins in the cell
...

The regulation of the lac operon;
 An operon was originally defined as a cluster of genes transcribed to produce
a single mRNA molecule from a promoter controlled by an operator (defined
as the binding site for a repressor)
 Later, found that a promoter/operator may control just a single gene
 The promoter for a gene or gene cluster may not be controlled by an
operator (e
...
only by one or more activators), or may not even be controlled
at all
...

 Operon is a mult-gene transcriptional unit (TU)

BLGY1232 Control of bacterial gene expression 2
Utilization of lactose

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3 genes are required for the utilization of lactose and are encoded into the lac
operon; lacZ, Y and A;
 -galactosidase (lacZ) -Cytoplasmic enzyme that breaks lactose into glucose
galactose
 Lactose permease (lacY) - An integral membrane protein that transports
lactose across cytoplasmic membrane
 Transacetylase (lacA) - May acetylate galactoside sugars (other than lactose),
preventing them becoming substrates for β galactosidase
Lac operon;
 Has a single promoter and terminator
 Promoter, operator, genes and terminator are considered part of the operon
(transcriptional unit)
 Although, all three genes are co-transcribed to make a single mRNA, each
gene is independently translated into protein from the same mRNA  This is
called a poly-cistronic or polygenic mRNA
 Simple system ensures all three genes are co-ordinately expressed
 Its regulator is found adjacent to it but the gene can be located somewhere
else in the chromosome as the regulator doesn’t have to be next to it
Controlling elements and genes;
 Gene expression is controlled at level of transcription initiation by an
adjacent gene called lacI
...

 lacI has its own promoter and terminator so is not part of the lac operon
 Control genes are not always adjacent to the cognate operon(s)
Order of genes;
 lacI: promoter-lacI-terminator
 operon: promoter-operator-lacZ-lacY-lacA-terminator
The catabolism of lactose;
 The specific activity (enzyme units per mg of total protein) of enzymes
required for glucose metabolism (e
...
via glycolysis) does not alter
significantly depending on the growth conditions
...
g
...


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 The specific activity of -galactosidase, which breaks lactose into glucose +
galactose, is ~1000-fold higher when cells are grown in lactose compared
with glucose, as sole carbon sources
...

 Following change from glucose to lactose as sole carbon source, up to 5,000
molecules of β-galactosidase can accumulate within minutes
...

Operons;
 An operon was originally defined as a cluster of genes transcribed to produce
a single mRNA molecule from a promoter controlled by an operator (defined
as the binding site for a repressor)
 Later, found that a promoter/operator may control just a single gene
 The promoter for a gene or gene cluster may not be controlled by an
operator (e
...
only by one or more activators), or may not even be controlled
at all
...

 However, many do not distinguish between operons and multi-gene
transcriptional units
...
coli grown in absence of lactose (presence
of glucose)  repressor proteins attach to operator and prevent transcription of
mRNA  RNA polymerase can bind to promoter when repressor is bound to
operator but cannot initiate transcription

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Functional state of the lac operon when E
...
In absence of lactose, residual levels of β-galactosidase (1-5 molecules per
cells) exist and these are able to isomerise lactose (when it becomes
available) to the allolactose
...
Allolactose is able to bind the lac repressor  This binding affects the
conformation of LacI
3
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4
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5
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6
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After induction;
1
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When cellular concentration of lactose decreases, the concentration of
allolactose also decreases
3
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4
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5
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6
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7
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coli to utilize
lactose efficiently are no longer being made (this will be qualified in the next
slide) by the cell!
8
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9
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The system is in equilibrium
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These β-galactosidase molecules are instrumental in the initial isomerisation
of lactose to allolactose that occurs when E
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The lac operon is said to be under negative control (LacI blocks RNA
polymerase, if inducer is absent)
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 The core of their model was that a response was mediated by regulating the
level of specific cellular proteins
...

 Now known that the activity levels of many enzymes (as well as nonenzymatic proteins) are not fixed and depend on the conditions used to grow
bacteria
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In fact, they
argued strongly that such mechanisms did not exist
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 When E
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Only when levels of glucose are depleted and growth slows is
the lac operon induced and lactose utilised
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coli is gown on lactose as the sole carbon source there is no repression and
there is activation which is absent in the presence of glucose;
 Low glucose levels stimulate the production of cAMP, this then binds to CAP
producing a conformational change that allows the CAP to bind DNA
...

 cAMP levels are related to the uptake of glucose

Catabolite repression and cAMP production;
 The cAMP level is related not to intracellular [glucose], but to the rate of
glucose uptake (by a phosphotransferase system); influences the activity of
membrane-associated adenylate cyclase
...

 Similar mechanisms for responding to external stimuli operate in eukaryotes
...

 CRP is able to bind its cognate sites only when bound to cAMP
 Transcription initiation by RNA holoenzyme is increased when its interacts on
the DNA with CRP
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Only results in high level of transcription
when lactose is present and repressor is not bound to its operator
 When there is a high level of cAMP it binds to the CAP (CRP) protein, which is
then able to bind a site upstream of the lac promoter
 CRP can then contact the RNA polymerase and this contact increases the
affinity of the RNA polymerase for the promoter
 In the absence of CAP (CRP) binding to the upstream site, transcription from
the lac promoter is lower
...

 When glucose and lactose are present, E
...

 Adding cAMP to cells restore transcription of the lac operon even when
glucose is still present
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 Protein-protein interactions can either repress (repressor-RNA polymerase)
or activate (CRP-RNA polymerase) transcription
 Small molecules can play key roles in gene expression by interacting with
regulatory proteins
 Genetic and biochemical studies are needed to identify and characterize the
interacting molecules - DNA and protein
 Allolactose, cAMP and tryptophan (see next) are examples of small molecules
that cause an allosteric change in a DNA-binding protein (i
...
they instruct a
change in protein conformation)
 Multiple signals are integrated at the level of a single promoter
...

 Provides examples of control other than at the level of transcription initiation
 Allosteric effects
 Phosphorylation
CPR is capable of activating the expression of more than 200 genes including those
for utilization of arabinose  an example of a global regulatory system as it binds to
more than one promoter  no regulatory system is identical to the lac operon;
There is great variety in both the modus operandii of the transcription factors and
their corresponding binding sites in the DNA (i
...
trans- and cis-acting components,
respectively)
e
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L-arabinose catabolism

 Also shows induction and catabolite repression; but in this case with regard to the
induction step binding of inducers such as L-arabinose converts the repressor into an
activator
Regulons;
 A regulon is defined as the entirety of all genes regulated positively
(enhanced transcription) and/or negatively (reduced transcription) by a
particular regulatory protein
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g
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 One of the best studied regulons is the SOS response, which is activated by
DNA damage and coordinates various functions to allow and mediated repair
 Central to the SOS (stimulus is internal and not related to nutrient availability
in environment) response is LexA which represses a number of genes 
activation of the SOS regulon;
1
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2
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3
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4
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coli and other bacteria
 Bacteria respond to changes in their environment by altering their pattern of gene
expression
 The most commonly found step that is control has been transcription initiation
...

 Gene expression also regulated at other levels; mRNA stability, translation, protein
stability, post-translational modifications
 Some genes are continuously expressed (al genes are regulated at some level), so
called housekeeping genes, such as those required for protein synthesis and glucose
metabolism

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