Transcript Document

Regulation of Transcription in Prokaryotes
•lac Operon
•ara Operon
Operon: A group of contiguous, coordinately
controlled genes.
A single mRNA molecule transcribed from
an operon contains multiple ORFs - called a
polycistronic message.
Transcriptional Regulation of Operons
•Regulatory sequences adjacent to an operon
determine whether it is transcribed or not.
•Regulatory sequences are primarily ‘operators’
(repressor binding sequences). Can also include
activator binding sequences.
•Regulatory proteins work with regulatory
sequences to control transcription of the operon.
Induction and Repression
•Induction refers to increased expression of genes in
response to a metabolite.
•Repression refers to decreased expression of genes in
response to a metabolite.
•IPTG, the artificial inducer of the lac operon, is an
example of a ‘gratuitous’ inducer: It cannot be
metabolized by b-galactosidase enzyme, but it still
induces b -galactosidase synthesis.
The lac operon allows the option of lactose
utilization as a carbon source
Genes Encoded by the lac Operon
•lacZ encodes b -galactosidase that cleaves lactose into
galactose and glucose, AND converts lactose to the
inducer allolactose.
•lacY encodes galactoside permease which is required
for transport of lactose into the cells.
•lacA encodes galactoside transacetylase which
transfers acetyl groups from acetyl-CoA to
b -galactosides. The biological function of the
enzymatic activity is unclear.
b -galactosidase Activity
1.
2.
β-galactosidase
lactose
b -1,4 linkage
allolactose
b-1,6 linkage
Regulation of the lac operon Involves:
Negative control
Positive control
Cis-acting sequence
Trans-acting factor
Negative control of the lac operon
Monod showed that Z+Y- mutants did not take up a radioactively
labeled galactoside in the presence of inducer. He concluded
that lacY must encode an enzyme responsible for transporting the
galactoside into cells: called it galactoside permease.
lac Genetics I
lac Genetics II
Jacob and Monod’s Operon Hypothesis
Based on Genetics
1. There are two key control elements of the operon:
the repressor gene and the operator to which the
repressor gene product binds.
2. There is a specific interaction between the inducer
and repressor that prevents the repressor from
binding to the operator.
3. All three lac genes are clustered under a single
control unit.
4. Subsequent deletion analysis showed that there is a
promoter necessary for the expression of all three
lac genes.
Biochemical studies confirm all of the tenets of
Jacob’s and Monod’s hypothesis.
Isolation of the lac Repressor
Assay for lac repressor activity: Binding to the
labeled synthetic inducer IPTG.
Problem: lac repressor is present in very tiny quantities
in the cell.
Solution: Isolate repressor from mutant E. coli strain
with lacIt mutation that causes repressor to bind to
IPTG more tightly than normal.
Prediction: If the protein isolated really was
the lac repressor, then it should bind to the lac operator in an
inducer sensitive manner (i.e. the addition of inducer should
prevent the repressor from binding to the operator).
Experiment: Cohn and colleagues used a nitrocellulose filter
binding assay. They mixed 32P-labeled lacO DNA with the protein
from above, and either added or left out IPTG.
Result: In the presence of protein and ABSENCE of IPTG the
labeled lacO DNA was retained on the filter.
Conclusion: The lac repressor had been isolated.
Also found the Oc operator bound with lower affinity to the
repressor as compared to the WT operator.
How does the lac repressor work?
1971 - Pastan and colleagues showed that RNA Pol
can bind tightly to the lac promoter and form an
open promoter complex in the presence of the lac
repressor.
1987 - Straney and Crothers showed that RNA Pol
and the lac repressor can bind simultaneously to
the lac promoter.
Hypothesis: The lac repressor prevents the transition
from transcription initiation to elongation.
Lee and Goldfarb Expt.
Expt: Run-off transcription assay.
1. Pre-incubate labeled DNA (contains lac control region
and beginning of lacZ gene) with (or without) repressor
for 10 min.
2. Add RNA Pol.
3. Add heparin, and all components of transc. rxn.
except CTP.
4. Add labeled [a-32P]CTP with or without IPTG.
Result of Lee and Goldfarb Expt.
Conclusion: RNA Pol forms an open
complex with the lac promoter even
in the presence of the lac repressor
in vitro. (sequence: promoter &DNA ,
repressor + or -, RNA pol, heparin,
all rxn components except CTP, then
alpha 32P CTP.
Observed that short abortive
transcripts are even shorter in the
presence of the repressor. Added
further support for the idea that the
repressor prevents the transition from
initiation to elongation.
Problem with Lee and Goldfarb Expt: Nonphysiological
concentrations of repressor, RNA Pol etc. were used.
(Concentrations were higher than in vivo.)
Record and Colleagues: Kinetic studies of effect of
lac repressor on dissociation of RNA Pol from promoter
in vitro using conditions closer to in vivo situation.
• Made RNA Pol/lac promoter complexes.
• Added or didn’t add heparin or lac repressor.
• Measured rate of abortive transcript synthesis by
including a fluorescently labeled UTP analog. Labeled
pyrophosphate was released when this analog was
incorporated into RNA.
Got paradoxical results!
Result of Record and colleagues Expt.
Results suggest that the lac repressor does prevent
binding of RNA Pol to the promoter!
There are 3 lac operators
All three operators are important for repression.
Structure of lac repressor tetramer bound to DNA
one dimer
of tetramer
operator
DNA
Positive control of the lac operon
CAP
+
protein
These are trans-acting factors.
Cis-acting sequence is activator
(or CAP) binding site.
cAMP signals low glucose
activator binding-site
lac operon off
low
lac operon very weakly on
lac operon fully induced
CAP -cAMP Activity
•CAP-cAMP complex recruits RNA Pol to the major
(P1) promoter of the lac operon to form closed
promoter complex
•lac operon has alternative promoter (P2)that is
inefficient. CAP-cAMP reduces initiation at P2
and stimulates initiation at P1.
CAP and RNA Pol bind cooperatively to DNA,
and physically interact.
Evidence:
1. CAP and RNA Pol cosediment in presence of cAMP.
2. When CAP and RNA Pol are both bound to lac control region,
they can be chemically cross-linked.
3. DNase footprinting shows that CAP and RNA Pol bind to
adjacent binding sites in DNA.
4. Certain mutations in CAP that reduce activation specifically
affect what is thought to be RNA Pol binding region of CAP.
(These same mutations don’t affect binding of DNA by CAP).
5. Deletion of aCTD of RNA Pol, region thought to bind to CAP,
prevents activation by cAMP-CAP.
6. In crystal structure, cAMP-CAP and aCTD touch
when cAMP-CAP and RNA Pol both bound to DNA.
Interaction of CAP-cAMP, RNA Pol and
DNA of lac control region
The ara Operon
•another example of operon that has both positive
and negative regulation
•araB, A, and D encode the 3 arabinose
metabolizing enzymes
•araC encodes the control protein AraC which is both
a positive regulator (in the presence of arabinose) and
a negative regulator (in the absence of arabinose).
•cAMP-CAP complex also acts as a positive regulator
Organization of the ara operon
Control of the ara Operon I - Negative
araPBAD
•When arabinose is absent, the AraC protein acts as
a negative regulator.
•AraC acts as a dimer, and causes the DNA to loop.
Looping brings the I1 and O2 sites in proximity to one
another.
•One AraC monomer binds to I1 and a second monomer
binds to O2.
•Binding of AraC prevents RNA Pol from binding to
the PBAD promoter
Control of the ara Operon II - Positive
araPBAD
•When arabinose is present, it binds to AraC and changes
AraC conformation
•An arabinose-AraC dimer complex binds preferentially
to I1 and I2, and NOT to O2 which causes ‘opening’
of the loop. This allows RNA Pol to bind to PBAD.
•If glucose levels are low, cAMP-CAP complex binds
to Pc.
•Active transcription occurs.
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Negative autoregulation of araC
transcription
•High levels of AraC cause the protein to bind to
O1 and inhibit transcription of the araC gene negative autoregulation.