Transcript Prokaryotic Regulation of Transcription

THE PROBLEM
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Prokaryotes must accomplish specialized
functions in one unspecialized cell
Options
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Have all gene products functioning at all times
(constitutive expression)
Turn on genes only as they are needed
(inducible expression)
Are examples of both types of expression
Control of Gene Function
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Control mRNA expression and lifetime
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Deviations from consensus promoter sequences
Activator proteins
UP elements
REMEMBER: prokaryotic mRNAs are
polycictronic, can have several genes involved
in a metabolic pathway expressed together
(coordinated expression)
Control translation and degradation of protein
product
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Half-life of protein
Position of cistron in polycistronic mRNA
Shine-Dalgarno deviations
Regulation (cont’d)
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Negative regulation—Protein (repressor)
inhibits transcription (Ex. LexA).
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Inducer– binds to repressor, alters form,
reduces affinity for target, allows expression
of gene.
Sometimes, small molecule required for
repressor activity.
Positive regulation—Activator
proteinincreases transcription rate.
Generally bound to a smaller signal
molecule.
Regulation of Enzyme Activity
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Degradation of
enzyme
Feedback
inhibition–
generally a form of
allosteric inhibition
Remember: the cell
is web of
competing
pathways.
The lac Operon
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Lactose—A disccharide hydrolyzed to
glucose and galactose .
Lactose metabolizing enzymes expresse as a
polycistronic message ( lacZ, lacY, lacA).
Is an inducible operon.
Consists of
 Regulatory components
 Structural components
The Players
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Regulatory
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Promoter (P)
Operator (O)
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LacI
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Structural
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lacZ
lacY
lacA
In The Absence of Lactose
Repressor tetramer
binds operator,
prevents
transcription
No reason for
expression
is repressed
In The Presence of Lactose
Conformational change
caused by inducer
reduces affinity of
repressor/inducer for
operator
Role of CRP·cAMP
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Expression of lac operon
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(+) Glucose (-) Lactose= No expression
(+) Glucose (+) Lactose= Low to no expression
(-) Glucose (+) Lactose= High expression
When [glucose] is high, [cAMP] is low and vice versa.
Cyclic AMP Receptor Protein forms a complex with
cAMP and binds at a site near the promoter.
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Strongly increases expression
Mechanism: causes bending of DNA, allows RNA pol 2 points
of caontact
CAP·cAMP Mechanism
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CAP-sensitive promoters usually weak
CAP·cAMP Bends DNA, allowing RNA pol to bind
at two points, stabilizing interaction
May also interact with C-terminal domain of
sigma
LEGEND:
Purple- CAP·cAMP
Red- RNA pol
Blue- Sigma
Galactose Operon
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Regulates catabolism of galactose
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3 cistrons encoding structural proteins
2 promoters (P1 and P2)
2 operators
Repressor (gal R)
Gal Operon Regulation
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Effect of cAMP levels
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CAP·cAMP regulates transcription from two promoters
in opposite ways
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CAP·cAMP activates from P1, inhibits from P2 when
[cAMP] transcribe from P1, when [cAMP] transcribes
form P2.
As long as no repression, level of Gal mRNA constant
Regulation
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Repressor- product of gal R
Inhibits from both operators
Galactose acts as inducer
If galactose absent, both promoters inactive
Gal Operon
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One unit of the
galR dimer binds to
each operator
Induces
conformational
change, prevents
transcription
Possible structures
Note: dimer responsible
for repression
Ara Operon
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Dual action regulatory
protein- AraC
 (-) arabinose
 Represses
 (+) arabinose
 Activates
AraI
 AraI1
 AraI2
Two operators
AraI
In absence of arabinose- AraC dimer causes loop by
joining I1 and O2. no transcription
Operators
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AraO1- regulates
AraC
AraO2- regulates
AraBAD
With arabinose, shape change causes dimer to
sit on I1 and I2, allowing transcription
Ara operon 2
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NOTE: CAP·cAMP binding site. Increases
transcription.
Autoregulation of AraC
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AraC transcribed from Pc.
Pc regulated by O1.
As level of AraC rises, binds to AraO1 and
prevents transcription from Pc.
prevents wasteful accumulation of repressor
Is an example of autoregulation
Are other models
Trp Operon
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Encodes enzymes necessary for Trp synthesis
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encodes a set of anabolic enzymes rather than
catabolic enzymes.
Anabolic enzymes are generally turned off by
presence of a product (feedback inhibition)
In addition to repression, system shows
attenuation, a finer level of control.
Structure
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5 structural genes3 enzymes
Promoter and operator precede structural genes
In absence of Trp, TrpR protein is inactive
Tryptophan Operon Repression
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Negative control of operon:
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Low tryptophan
No repression
transcription
Positive control of operon:
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High tryptophan
Tryptophan (a corepressor) combines with
free repressor dimer (aporepressor
dimer)=repressor dimer
transcription blocked
Attenuation: A Finer Level of Control
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Trp operon expression also regulated by
attenuation, a much finer level of control.
Trp operon features
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Repression very weak
transcription could occur even in presence of
repressor
 Very energy expensive
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Attenuation increases expression 10-fold
Result: Trp operon expression spans a 700fold range (from inactive to fully active)
Attenuation Mechanism
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Special sequences prior between promoter and
structural gene
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Trp leader
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Trp attenuator
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Has translation start site
2 Trp codons in a row (very rare)
Has transcription termination sequence
These sequences weaken (attenuate)
transcription when trp is abundant
Operates by causing premature termination of
transcription
REMEMBER: transcription and translation occur
simultaneously in prokaryotes
Attenuation Mechanism 2
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Different hairpin configurations
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Configuration 1—Two hairpins, 4 stems
Configuration 2- One hairpin, two stems
Configuration 1 is more stable
Translation begins as soon as Trp leader
transcript emerges
If Trp is in short supply
Ribosome will stall over Stem 1
If Trp Abundant
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Ribosome translates,
hits termination
codon, falls off
Allows formation of 2
hairpins
One contains intrinsic
terminator
RNA pol falls off
If Trp is Scarce- Overriding
Attenuation
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Ribosome will stall
over Trp codons in
Trp leader sequence
Double hairpin can’t
form, only single
hairpin configuration
Allows RNA pol to
transcribe through
termination
sequences
The Operons
OPERON INDUCER Repressor CAP COMMENTS
gene
One promoter, one operator
lac
lactose
lacI
+
gal
galactose galR
+
Two operators, 2 promoters
ara
arabinose araC
+
araC protein acts as repressor
and activator
Trp
Low Trp
-
trpR
(aporepressor)
Anabolic, Trp is
corepressor, attenuation