Transcript Section L

Section L: Regulation of transcription
in prokaryotes
L1 The lac operon
L2 The trp operon
L3 Transcriptional regulation
by
alternative σ factors
L1:The LAC Operon
Operon - what is it?
The operon is a unit of gene expression and
regulation
•
The structural genes (any gene other than a regulator) for
enzymes involved in a specific biosynthetic pathway whose
expression is co-ordinately controlled.
•
Control elements such as an operator sequence, which is a
DNA sequence that regulates transcription of the structural
genes.
•
Regulator gene(s) whose products recognize the control
elements, for example a repressor which binds to and
regulates an operator sequence.
The structure of operon
Francois Jacob and Jacques Monod
(Pasteur Institute, Paris, France)
•
Studied the organization and control of the lac operon in E. coli.
•
Earned Nobel Prize in Physiology or Medicine 1965.
E. coli’s lac operon
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E. coli expresses genes for glucose metabolism continuously.
•
Metabolism of other alternative types of sugars (e.g., lactose) are
regulated specifically.
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Lactose = disaccharide (glucose + galactose), provides energy.
•
Lactose acts as an inducer (effector molecule) and stimulates
expression of three proteins at 1000-fold increase:
•-galactosidase (lacZ)
An enzyme responsible for hydrolysis of lactose to galactose
and glucose .
•Permease (lacY)
An enzyme responsible for lactose transport across the
bacterial cell wall.
•Acetylase (lacA) Function is not understood.
-galactosidase and structure of lactose
E. Coli cells need an enzyme to break the lactose down into
its two component sugars: galactose and glucose. The
enzyme that cuts it in half is called  -galactosidase.
Structure of the lac operon
Lac operon
-galactosidase Permease
Acetylase
DNA
lacI:
promoter-lacI-terminator
operon:
promoter-operator-lacZ-lacY-lacA-terminator
Without inducer-no structure genes expression
Regulation genes
Lac operon
-galactosidase
Permease Acetylase
DNA
transcript
No structure genes expression
mRNA of repressor
translate
Inactive lac repressor
Binding of inducer inactivates the lac repressor
cAMP receptor protein
The Plac promoter is not a strong
promoter. Plac and related
promoters do not have strong -35
sequences and some even have
weak -10 consensus sequences.
For high level transcription, they
require the activity of a specific
activator protein called cAMP
receptor protein (CRP). CRP may
also be called catabolite activator
protein or CAP.
Glucose reduces the level of cAMP
in the cell. When glucose is absent,
the levels of cAMP in E. coli
increase and CRP binds to
cAMP.So, the CRP-cAMP complex
binds to the lactose operon.
DNA-bending and Transcription
regulation
CAP-cAMP binding to the lac activator-binding site recruits RNA
polymerase to the adjacent lac promoter to form a closed promoter
complex. This closed complex then converts to an open promoter
complex. CAP-cAMP bends its target DNA by about 90° when it
binds. And this is believed to enhance RNA polymerase binding to
the promoter, enhancing transcription by 50-fold.
L2 The TRP operon
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If amino acids are present in the growth medium E. coli will
“import” amino acids before it makes them, genes for
amino acid synthesis are repressed.
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When amino acids are absent in the growth medium, genes
are “turned on” (or expressed) and amino acid synthesis
occurs.
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The tryptophan (Trp) operon of E. coli is one of the most
extensively studied operons in amino acids synthesis.
first characterized by Charles Yanofsky et al.
Structure of the trp operon and function of the trp repressor
transcription stop site
attenuator
trpR
Active trp
repressor
Trp repressor
Enzyme for tryptophan synthesis
tryptophan
A gene product of the separate trpR operon, the trp repressor, specifically interacts
with the operator site of the trp opseron. The symmetrical operator sequence, which
forms the trp repressor-binding site, overlaps with the trp promoter sequence
between bases -12 and +3.
The attenuator
At first, it was thought that the repressor was responsible for all of
the transcriptional regulation of the trp operon.
However, it was observed that the deletion of a sequence between
the operator and the trpE gene coding region resulted in an
increase in both the basal and the activated level if transcriptio.
This site is termed the attenuator and it lies towards the end of
transcribed leader sequence of 162 nt that precedes the trpE
initiator codon.
The attenuator is a rho-independent terminator site which has a
short GC-rich palindrome followed by eight successive U residues.
If this sequence is able to form a hairpin structure in the RNA
transcript, then it acts as highly efficient transcription terminator
and only a 140bp transcript is synthesized.
Leader RNA structure and leader peptide
14aa
hairpins
Pause
Anti-termination
Termination
Molecular model for attenuation (cont.):
Position of the ribosome plays an important role in attenuation:
When Trp is scarce or in short supply (and required):
1.
Trp-tRNAs are unavailable, ribosome stalls at Trp codons and
covers attenuator region 1.
2.
Region 1 cannot pair with region 2, instead region 2 pairs
with region 3 when it is synthesized.
3.
Region 3 (now paired with region 2) is unable to pair with
region 4 when it is synthesized.
4.
RNA polymerase continues transcribing region 4 and beyond
synthesizing a complete trp mRNA.
Molecular model for attenuation (cont.)
Position of the ribosome plays an important role in attenuation:
When Trp is abundant (and not required):
1.
Ribosome does not stall at the Trp codons and continues
translating the leader polypeptide, ending in region2.
2.
Region 2 cannot pair with region 3, instead region 3 pairs
with region 4.
3.
Pairing of region 3 and 4 is the “attenuator” sequence and
acts as a termination signal.
4.
Transcription terminates before the trp synthesizing genes
are reached.
Importance of attenuation
• The presence of tryptophan gives rise to a 10-fold repression
of trp operon transcription through the process of attenuation
alone.
• Combined with control by the trp repressor (70-fold), thus
means that tryptophan levels exert a 700-fold regulatory effect
on expression from the trp operon.
•Attenuation occurs in at least six operons that encode enzyme
concerned with amino acid biosynthesis.
L3 Transcriptional regulation by
alternativeσfactors
•σ factors appear to be bifunctional proteins that stimultaneously can
bind to core RNA polymerase and recognize specific promoter sequence in
DNA.
• Many bacteria, including E. coli, produce a set ofσfactors that recognize
different sets of promoters.
•Some environmental conditions require a massive change in the overall
pattern of gene expression in the cell.
•Under such circumstances, bacteria may use a different set of σ factors to
direct RNA polymerase binding to different promoter sequences.
Promoter recognition
The binding of an alternative σfactors to RNA polymerase can confer a
new promoter specificity on the enzyme responsible for the general RNA
synthesis of the cell.
Comparisons of promoters activated by polymerase complexed to specific
σfactors show that each σfactor recognizes a different combination of
sequences centered approximately around the -35 and -10 sites. It seems
likely that σfactors themselves contacet both of these regions, with the -10
region being most important.
The σ70 subnuit is the most common σfactor in E. coli which is responsible
for recognition of general promoters which have consensus -35 and -10
elements.
Heat shock promoter
Comparison of the heat-shock (σ32) and general (σ70) responsive promoters
When E. coli is subjected to an increase in temperature, the synthesis of
a set of around 17 proteins, called heat-shock proteins, is induced. The
promoters for E. coli heat-shock proteins-encoding genes are recognized
by a unique form of RNA polymerase holoenzyme containing a variant
σfactor σ32, which is encoded by the rpoH gene.
Sporulation in Bacillus subtilis
Vegetatively growing B.subtilis cells from bacterial spores(see Topics
A1) in response to a sub-optimal environment.
The RNA polymerase in B.subtilis is functionally identical to that in
E.coli. The vegetatively growing B.subtilis contains a diverse set of
σfactors.
Sporulation is regulated by a further set of σfactors in addition to
those of the vegetative cells.
Different σfactors are specifically active before cell partition occurs,
in the forespore and in the mother cell. Cross-regulation of this
compartmentalization permits the forespore and mother cell to
tightly co-ordinate the differentiation process.
Bacteriophage σfactors
Some bacteriophages provide new σsubunits to endow the host RNA
polymerase with a different promoter specificity and hence to selectively
express their own phage genes(e.g. phage T4 in E.coli and SPO1 in B.subtilis).
This stragety is an effective alternative to the need forfor the phage to encode
its own complete polymerase(e.g. bacteriophage T7,see Topic K2).
The B.subtilis bacteriophage SPO1 expresses a ‘cascade’ of σfactors in
sequence to allow its own genes to be transcribed at specific stage during
virus infection. Initially, early genes are expressed by normal bacterial
holoenzyme.
Among these early genes is the gene encoding σ28, which then displaces the
bacterial σfactor from the RNA polymerase.
The σ28-containing holoenzyme is then responsible for expression of the
middle genes. The phage middle genes include genes 33 and 34 which
specificy a further σ factor that is responsible for the specific trancription of
late genes. In this way, the bacteriophage uses the host’s RNA polymerase
machinery and expresses its genes in a defined sequential order.