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 of gene expression

Regulation
of Gene Expression
RNA polymerase
Regulatory proteins aa-tRNAs

DNA

Promoter Attenuator
operator
Transcription

RNA polymerase

Stop signal
Transcriptional control
(a) Transcription initiation:
positive/negative
(b) Transcription termination:
attenuation/anti-termination

Regulatory proteins
Antisense RNAs

mRNA
Ribosome
binding
site
Translation
Degradation

Stop signal
Translational control
Translation initiation:
positive/negative

Protein

Post-translational control
(e.g., proteolysis)

Regulation of gene expression

Regulation
of Gene Expression
RNA polymerase
Regulatory proteins aa-tRNAs

DNA

Promoter Attenuator
operator
Transcription

RNA polymerase

Stop signal
Transcriptional control
(a) Transcription initiation:
positive/negative
(b) Transcription termination:
attenuation/anti-termination

Regulatory proteins
Antisense RNAs

mRNA
Ribosome
binding
site
Translation
Degradation

Stop signal
Translational control
Translation initiation:
positive/negative

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

5ppp

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

O OH

OH

OH

OH

Lactose
-Galactosidase
HOCH2
O O
HO
OH

CH2
HO

O OH

OH
OH

OH

Allolactose
-Galactosidase
HOCH2

O OH

OH
OH

Galactose

HOCH2

HO

O OH

OH
OH

Glucose

The lac operon

Pi lacI

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

Whenlactoseispresent,itactsasaninduceroftheoperon.Itenters
thecellandbindstotheLacrepressor,inducingaconformational
changethatallowstherepressortofallofftheDNA.NowtheRNA
polymeraseisfreetomovealongtheDNAandRNAcanbemadefrom
thethreegenes.Lactosecannowbemetabolized.

Remember, the
repressor acts
as a tetramer

Whentheinducer(lactose)isremoved,therepressorreturnstoits
originalconformationandbindstotheDNA,sothatRNApolymerase
cannolongergetpastthepromotertobegintranscription.NoRNAand
noproteinaremade.

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

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

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

RNAP

cAMP
Inactive
CAP
CAP-cAMP
Active CAP

How does glucose reduce cAMP level?

O

O Adenine
H H C
C C H
Glucose
OH OH

- -

C
H

- -

ATP

- -

-O-P~O-P~O-P-O-CH

Adenylate
cyclase

- -

cAMP

H
C
O

H C
C H
OH

- -

C
H

O Adenine

- -

O=P
O-

- -

O-CH2

OUT

IN

IIAGlc-P

- -

= -

= -

= -

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 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 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

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

lacZ

lacY

lacA

(b) Glucose present (cAMP low); lactose present

Pi lacI

P
X

X
mRNA

mRNA

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

lacZ

lacY

lacA

(b) Glucose present (cAMP low); lactose present

Pi lacI

P
X

X
mRNA

mRNA

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

DNA

Promoter Attenuator
operator
Transcription

RNA polymerase

Stop signal
Transcriptional control
(a) Transcription initiation:
positive/negative
(b) Transcription termination:
attenuation/anti-termination

Regulatory proteins
Antisense RNAs

mRNA
Ribosome
binding
site
Translation
Degradation

Stop signal
Translational control
Translation initiation:
positive/negative

Protein

Post-translational control
(e.g., proteolysis)

Transcription termination players:

Termination sequence
RNA polymerase
and sometimes the Rho ( factor
RNAP

A
Promoter

Operon of 4 genes

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

CCCAUAGACUAACGAAAUGCGUACCACUUAUGUGACGGGCAAAG

A 3
2
GCCCGCCUAAUGAGCGGGCUUUUUUUUGAACAAAAUUAGAGA-3

mRNA forms secondary structures

1

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