Scotty Merrell
Department of Microbiology and Immunology
B4140
dmerrell@usuhs.mil
Regulation of Gene Expression I
QUESTIONS
1.
Why does the expression of genes need to
be regulated?
2.
Why is it important to study gene regulation?
3.
How is the expression of genes regulated?
4.
How do we study gene regulation?
Bacteria experience different conditions depending on environment
Pathogenic bacteria:
External reservoir
Host
Infection site #1
Infection site #2
QUESTIONS
1.
Why does the expression of genes need to
be regulated?
2.
Why is it important to study gene regulation?
3.
How is the expression of genes regulated?
4.
How do we study gene regulation?
Pathogenic bacteria produce virulence factors when
they sense they are inside of a host
ICDDR,B
Vibrio cholerae, the cause of cholera, produces toxin inside
of the host. Understanding regulation of expression of this toxin
is a means of understanding ways to prevent its production.
QUESTIONS
1.
Why does the expression of genes need to
be regulated?
2.
Why is it important to study gene regulation?
3.
How is the expression of genes regulated?
4.
How do we study gene regulation?
Regulation
gene expression
Regulation
of ofGene
Expression
RNA polymerase
Regulatory proteins aa-tRNAs
RNA polymerase
DNA
Promoter Attenuator
operator
Transcription
Stop signal
Transcriptional control
(a) Transcription initiation:
positive/negative
(b) Transcription termination:
attenuation/anti-termination
Regulatory proteins
Antisense RNAs
mRNA
Ribosome
binding
site
Translation
Stop signal
Translational control
Translation initiation:
positive/negative
Degradation
Protein
Post-translational control
(e.g., proteolysis)
Regulation
gene expression
Regulation
of ofGene
Expression
RNA polymerase
Regulatory proteins aa-tRNAs
RNA polymerase
DNA
Promoter Attenuator
operator
Transcription
Stop signal
Transcriptional control
(a) Transcription initiation:
positive/negative
(b) Transcription termination:
attenuation/anti-termination
Regulatory proteins
Antisense RNAs
mRNA
Ribosome
binding
site
Translation
Stop signal
Translational control
Translation initiation:
positive/negative
Degradation
Protein
Post-translational control
(e.g., proteolysis)
Transcription initiation

TTGACA
AACTGT
-37
-30
-35 region
TATAAT
ATATTA
-13
-6
-10 region
+1
mRNA
Promoter
 

’

Holoenzyme
Polymerase binds to
promoter region, forming
a closed complex
 

 ’
Polymerase unwinds
DNA, forming an
open complex

 

5’ppp
’
Core enzyme
Transcription begins
RNA polymerase-promoter interactions
Some promoters contain UP elements that stimulate transcription
through direct interaction with the C-terminal domains of the
 subunits of the RNA polymerase
Arrangement of  subunits on UP elements
Promoter with a full UP
element containing two
consensus subsites.
Promoter with an UP
element containing only a
consensus proximal
subsite.
Promoter with an UP
element containing only a
consensus distal subsite.
Genes come in two main flavors:
1. Constitutively expressed (transcription initiation
is not regulated by accessory proteins)
2. Regulated (transcription initiation
is regulated by accessory proteins)
a. Negatively Regulated--Repressor Protein
b. Positively Regulated--Activator Protein
Mechanisms of Regulation of Transcription Initiation:
Negative Regulation
RNA Polymerase
Mechanisms of Regulation of Transcription Initiation:
Negative Regulation
Repressor
Co-repressor
Repressor
Inactivator
Repressor
The lac operon
a model for negative regulation
A bacterium's prime source of food is glucose, since it does
not have to be modified to enter the respiratory pathway. So
if both glucose and lactose are around, the bacterium wants to
turn off lactose metabolism in favor of glucose metabolism.
There are sites upstream of the lac genes that respond to
glucose concentration.
This assortment of genes and their regulatory regions is called
the lac operon.
HOCH2
O
HO
OH
HOCH2
O OH
O
OH
OH
OH
Lactose
-Galactosidase
HOCH2
O O
HO
OH
CH2
HO
O OH
OH
OH
OH
Allolactose
-Galactosidase
HOCH2
HOCH2
O OH
OH
OH
Galactose
+
O OH
HO
OH
OH
Glucose
The lac operon
Pi lacI
P
O
lacZ
lacY
lacA
Structural genes:
lacZ encodes -galactosidase
lacY encodes -galactoside permease
lacA encodes -galactoside transacetylase
Regulatory gene and elements:
lacI --- encodes repressor protein
lacO --- operator
lacP --- promoter
The lac promoter and operator regions
The Lac Repressor is constitutively expressed
Lac Repressor
(monomer)
(tetramer)
Repressor binding
prevents transcription
When lactose is present, it acts as an inducer of the operon. It enters
the cell and binds to the Lac repressor, inducing a conformational
change that allows the repressor to fall off the DNA. Now the RNA
polymerase is free to move along the DNA and RNA can be made from
the three genes. Lactose can now be metabolized.
Remember, the
repressor acts
as a tetramer
When the inducer (lactose) is removed, the repressor returns to its
original conformation and binds to the DNA, so that RNA polymerase
can no longer get past the promoter to begin transcription. No RNA and
no protein are made.
Remember, the
repressor acts
as a tetramer
How to identify the regulatory elements?
1. Mutation in the regulatory circuit may either abolish
expression of the operon or cause it to occur without
responding to regulation.
2. Two classes of mutants:
A. Uninducible mutants: mutants cannot be expressed at all.
B. Constitutive mutants: mutants continuously express
genes that do not respond to regulation.
3. Operator (lacO): cis-acting element
Repressor (lacI): trans-acting element
Definitions:
cis-configuration: description of two sites on the same
DNA molecule (chromosome)
or adjacent sites.
cis dominance: the ability of a gene to affect genes
next to it on the same DNA molecule
(chromosome), regardless of the nature
of the trans copy. Such mutations exert
their effect, not because of altered
products they encode, but because of a physical
blockage or inhibition of RNA transcription.
trans-configuration:description of two sites on different
DNA molecules (chromosomes)
or non-contiguous sites.
Constitutive mutants:
do not respond to regulation.
Mutations that inactivate the lacI gene (lacI-)
cause the operon to be constitutively
expressed, because the mutant repressor
protein cannot bind to the operator.
Pi lacI- P O
mRNA
lacZ
X
lacY
lacA
mRNA
Nonbinding
repressor
Would this be a cis-dominant or recessive mutation?
Constitutive mutants can be recessive
Constitutive mutants in the lacI gene are recessive
Pi lacI- P O
mRNA
Pi lacI+
mRNA
lacZ
lacY
lacA
Constitutive mutants can also be dominant if the mutant allele
produces a “bad” subunit, which is not only itself unable to bind to
operator DNA, but is also able to act as part of a tetramer to prevent
any “good” (wild type LacI) subunits from binding.
Pi lacI- P O
mRNA
lacZ
X
lacI+
lacA
mRNA
et al.
mRNA
lacY
Think about how you could determine
whether a mutation was dominant or
recessive.
Questions about negative
Regulation of lac ?
Mechanisms of Regulation of Transcription Initiation:
Positive Regulation
RNA Polymerase
Mechanisms of Regulation of Transcription Initiation:
Positive Regulation
RNA Polymerase
Activator
The lac operon
a model for positive regulation
When levels of glucose (a catabolite) in the cell are high, a
molecule called cyclic AMP is inhibited from forming. So
when glucose levels drop, more cAMP forms. cAMP binds to
a protein called CAP (catabolite activator protein), which is
then activated to bind to the CAP binding site. This activates
transcription, perhaps by increasing the affinity of the site for
RNA polymerase. This phenomenon is called catabolite
repression, a misnomer since it involves activation, but
understandable since when it was named, it seemed that
the presence of glucose repressed all the other sugar
metabolism operons.
CAP --- a positive regulator
1. Catabolite repression: the decreased expression of many
bacterial operons that results from addition of glucose. Also
known as “glucose effect” or “glucose repression”.
2. E. coli catabolite gene activator protein (CAP; also
known as CRP, the cAMP receptor protein).
3. CAP-cAMP activates more than 100 different promoters,
including promoters required for utilization of alternative
carbohydrate carbon sources such as lactose, galactose,
arabinose, and maltose.
CAP --- a positive regulator
A. under catabolite-respressing conditions
cAMP level is very low
crp
Inactive CAP
Target operon
cAMP
B. Under non-catabolite-respressing conditions
cAMP level is very high
crp
Target operon
RNAP
cAMP
Inactive
CAP
CAP-cAMP
Active CAP
Activation
Autoregulation
RNAP
How does glucose reduce cAMP level?
O
O
O
O
O
H H C
C C H
OH OH
- -
C
H
Adenylate
cyclase
- -
cAMP
H
C
O
H C
C H
OH
- -
C
H
O Adenine
- -
O=P
O--
- -
O-CH2
IN
OUT
O Adenine
2
- -
ATP
- -
-O-P~O-P~O-P-O-CH
IIAGlc-P
- -
= -
= -
= -
O
Glucose
PTS
Glucose-6-P
IIAGlc
PTS - phosphoenolpyruvate-dependent carbohydrate
phosphotransferase system
Glc
IIA - glucose-specific IIA protein, one of the
enzymes involved in glucose transport.
1. IIAGlc-P activates adenylate cyclase.
2. Glucose decreases IIAGlc-P level,
thus reducing cAMP production.
3. Glucose also reduces CAP level:
crp gene is auto-regulated by
CAP-cAMP.
Activation of expression of the lac operon
E. coli CAP (CRP) --- 209 amino acids
Dimerization and cAMP-binding
DNA-binding
Helix-turn-helix
1-139
140-209
AR1
NH2-
156-164
His19
His21
Glu96
Lys101
AR2
-COOH
Transcription activation by CAP at class I
CAP-dependent promoters
(-62)
Transcription activation:
1. Interaction between the AR1 of the downstream CAP subunit and one copy of CTD.
2. The AR1-CTD interaction facilitates the binding of CTD to the DNA downstream of CAP.
3. Possibly, interaction between same copy of CTD and the 70 bound at the –35 element.
4. The interaction between the second CTD and CAP is unclear.
The result: increasing the affinity of RNAP for promoter DNA, resulting in an
increase in the binding constant KB, for the formation of the RNAP-promoter closed complex
Transcription activation by CAP at class I
CAP-dependent promoters (cont.)
(-103, -93, -83, or –72)
Transcription activation:
Possibly, the second copy of CTD may interact with the DNA downstream of CAP, and
may interact with the 70 bound at the –35 element.
Results: increasing the affinity of RNAP for promoter DNA, resulting in an
increase in the binding constant KB, for the formation of the RNAP-promoter closed complex
Transcription activation by CAP at class II
CAP-dependent promoters (cont.)
(-42)
Transcription activation:
1. Interaction between the AR1 of the upstream CAP subunit and one copy of CTD
(either CTDI or CTDII, but preferentially CTDI). The AR1-CTD
interaction facilitates the binding of CTD to the DNA upstream of CAP.
Results: increase in the binding constant KB, for the formation of the RNAP-promoter
closed complex
2.
Interaction between the AR2 of the downstream CAP subunit and NTDI.
Result:
increase the rate constant, kf, for isomerization of closed complex to open complex.
Transcription activation by CAP at class III
CAP-dependent promoters
(-103 or –93)
(-62)
Transcription activation:
Each CAP dimer functions through a class I mechanism with AR1 of the
downstream subunit of each CAP dimer interacting with one copy of CTD
Results: synergistic transcription activation
Transcription activation by CAP at class III
CAP-dependent promoters (cont.)
(-103, -93, or -83)
(-42)
Transcription activation:
The upstream CAP dimer functions by a class I mechanism, with AR1 of the
downstream subunit interacting with one copy of CTD; the downstream CAP
dimer functions by a class II mechanism, with AR1 and AR2 interacting with the
other copy of CTD and NTD, respectively.
Results: synergistic transcription activation
(a) Glucose present (cAMP low); no lactose;
P
Pi lacI
O
lacZ
lacY
lacA
(b) Glucose present (cAMP low); lactose present
Pi lacI
P
X
X
mRNA
mRNA
O
lacZ
lacY
lacA
No lactose inside the cells!
(inducer exclusion)!
Repressor Repressor
monomer tetramer
Repressor Repressor
monomer tetramer
(c) No glucose (cAMP high); lactose present
High level
of mRNA
X
mRNA
cAMP
CAP
Repressor
monomer
Inactive
repressor
Inducer
Repressor
tetramer
High
Glucose effect on
the E. coli lac operon
(a) Glucose present (cAMP low); no lactose;
P
Pi lacI
O
lacZ
lacY
lacA
(b) Glucose present (cAMP low); lactose present
Pi lacI
P
X
X
mRNA
mRNA
O
lacZ
lacY
lacA
No lactose inside the cells!
(inducer exclusion)!
Repressor Repressor
monomer tetramer
Repressor Repressor
monomer tetramer
(c) No glucose (cAMP high); lactose present
High level
of mRNA
X
mRNA
cAMP
CAP
Repressor
monomer
Inactive
repressor
Inducer
Repressor
tetramer
High
Glucose effect on
the E. coli lac operon
Inducer exclusion: How does it work?
1. Uptake of glucose
dephosphorylates
enzyme IIglc.
2. Dephosphorylated
enzyme IIglc binds to and
inhibits lactose permease.
3. Inhibition of lactose
permease prevents
lactose from entering the
cell.
4. Hence, the term inducer
exclusion.
Questions about positive regulation
of the lac operon?
Dual positive and negative control
of transcription initiation:
the E. coli ara operon
The E. coli L-arabinose operon
+
+
AraC exists in two states
Arabinose
P1
P2
Antiactivator
Activator
Arabinose
AraC acts as a positive or negative regulator based
on its conformational state and binding affinity for
various sites in the two promoter regions.
AraC encodes the regulator
AraO1 and AraO2 encode operators
CAP is a CAP binding site
AraI is an additional regulatory region
AraBAD are the structural genes
In the absence of arabinose, the P1 form of AraC binds AraO2 and
AraI to prevent any P2 form from binding and activating expression
--this is anti-activation, not repression!
No arabinose
+ arabinose
In the presence of arabinsose, AraC shifts to the P2 form and binds
AraI and acts to activate transcription.
If AraC concentration becomes too high, AraC will also bind to AraO1
and repress its own expression.
Therefore AraC is an Activator, Repressor and Anti-activator!!
The regulatory regions of the PC and PBAD promoters
The domain structure
of one subunit of the
dimeric AraC protein
The PC and PBAD Regions in the presence
or absence of arabinose
+ L-arabinose
Hypothetical model of the activation of the PBAD promoter
1. PBAD – class II promoter
2. Possible interactions: between the CTD of RNAP
and the CAP protein and AraC protein and DNA
Strategies for Understanding Regulation
1. Find mutations that render the regulation uninducible or constitutive.
2. Decide by performing a complementation test if the mutants are dominant or
recessive.
3. If they are recessive, decide if the system is regulated by repression or by
activation. A recessive mutated activator has most likely lost function: the
system will become uninducible. A recessive mutated repressor has also lost
function, but now the system will show constitutive expression.
4. Decide if the elements of the system act in cis or in trans to each other: are
they proteins or DNA binding sites?
5. Construct a model.
Questions about ara regulation?
Regulatory mechanisms used to control gene expression
A. Transcriptional control
1. Transcription initiation
a) Positive
b) Negative
2. Transcription termination
Attenuation
B. Translational control
1. Positive
2. Negative
C. Post-translational control--Proteolysis
Regulation of gene expression
RNA polymerase
Regulatory proteins aa-tRNAs
RNA polymerase
DNA
Promoter Attenuator
operator
Transcription
Stop signal
Transcriptional control
(a) Transcription initiation:
positive/negative
(b) Transcription termination:
attenuation/anti-termination
Regulatory proteins
Antisense RNAs
mRNA
Ribosome
binding
site
Translation
Stop signal
Translational control
Translation initiation:
positive/negative
Degradation
Protein
Post-translational control
(e.g., proteolysis)
Transcription termination players:
Termination sequence
RNA polymerase
and sometimes the Rho (r) factor
RNAP
A
Promoter
B
C
Operon of 4 genes
D
X
Terminator
Two major types of Terminator Sequences
1. Rho-independent
2. Rho-dependent
Rho-independent
terminator
Rho-independent
terminator
Rho-dependent
terminator
Attenuation:
Premature termination of transcription
of operons for amino acid biosynthesis
(trp, his, leu, etc.)
Relies on coupled transcription and translation and
RNA secondary structure
Organization of Tryptophane Biosynthesis Genes
P/O
trpR
P/O L trpE trpD trpC
trpB trpA
mRNA
mRNA
Tryptophan
repressor
End product of the pathway
The trp leader mRNA encodes the LEADER PEPTIDE
MetLysAlaIlePheValLeuLysGlyTrpTrpArgThrSer
5’-AUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCC
U
1
CCCAUAGACUAACGAAAUGCGUACCACUUAUGUGACGGGCAAAG
A 3
2
GCCCGCCUAAUGAGCGGGCUUUUUUUUGAACAAAAUUAGAGA-3’
4
mRNA forms secondary structures
1
2
3
4
Pre-emptor
3 and 4 form a
Rho-independent
terminator
2 and 3 form the
Two possible alternative structures can form Pre-emptor, which prevents
2 is complementary to 1 and 3
Terminator formation
3 is complementary to 2 and 4
Adapted from http://www.andrew.cmu.edu/user/berget/Education/attenuation/atten.html
Tryptophan absent
Tryptophan present
UGGUGGCGCACUUCCU
UGGUGGCGCACUUCCU
Biosynthetic Operons Regulated by Attenuation
Operon
his
pheA
leu
thr
ilv
Leader Peptide Sequence
Regulatory
Amino Acid(s)
Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His
-His-His-His-Pro-Asp
His
Met-Lys-His-Ile-Pro-Phe-Phe-Phe-Ala-Phe-Phe
-Phe-Thr-Phe-Pro
Phe
Met-Ser-His-Ile-Val-Arg-Phe-Thr-Gly-Leu-Leu
-Leu-Leu-Asn-Ala-Phe-Ile-Val-Agr-Gly-Agr-Pro
-Val-Gly-Gly-Ile-Gln-His
Leu
Met-Lys-Agr-Ile-Ser-Thr-Thr-Ile-Thr-Thr-Thr
-Ile-Thr-Ile-Thr-Thr-Gly-Asn-Gly-Ala-Gly
Thr, Ile
Met-Thr-Ala-Leu-Leu-Arg-Val-Ile-Ser-Leu-Val
-Val-Ile-Ser-Val-Val-Val-Ile-Ile-Ile-Pro-Pro
-Cys-Gly-Ala-Ala-Leu-Gly-Arg-Gly-Lys-Ala
Leu, Val, Ile
Attenuation can also occur at the level of
Protein-RNA interaction:
Regulation of the trp operon in Bacillus
Model of trp
transcriptional
control
Binding of
activated TRAP
in the leader
peptide
results in the
formation of a
terminator
structure
Take home message:
Transcription of genes to produce mRNA
can be controlled at the level of
initiation and/or termination
STOP