GENE REGULATION IN PROKARYOTES

Repression of lac gene expressionIn the absence of lactose, repressor binds to the operator and prevents transcription lac repressor is a negative regulatory element General Genetics-BIO

7TH WEEK, BIO-1053

GENE REGULATION IN PROKARYOTES

General Genetics-BIO1053

7 th week

Histochemical localization of GUS expression under control

of the Atndt1 and Atndt2 promoter in A thaliana.

Hierarchical clustering of top-500 most variable genes

General Genetics-BIO1053

7 th week

Chapter outline

Overview of Prokaryotic Gene Regulation

The Regulation of Gene Transcription

Attenuation of Gene Expression: Termination of Transcription

Global Regulatory Mechanisms: E.coli’s response

to heat shock

The regulation of gene expression can occur

at many steps

Transcriptional control

• Binding of RNA polymerase to promoter

• Most critical step in regulation of most prokaryotic genes

• Shift from initiation to elongation

• Constitutive

– Synthesized all the time in fixed amounts

– Does not respond to the environment

• Adaptive

– Responds to the environment

• Inducible

• Repressible

• Induction: stimulation of protein synthesis

• Inducer: molecule responsible for induction

• Repression: inhibition of protein synthesis

response to an inducer, usually

the enzyme’s substrate

Repressible – Repressed by the

co-repressor, usually the product

end-Operon: Group of genes transcribed into a single mRNA

Includes promoter & operator sequences

The lactose operon in E coli

The players

• Three structural genes - lacZ, lacY, and lacA

• Promoter - site to which RNA polymerase binds

• Cis-acting operator site – controls transcription initiation

• Trans-acting repressor - binds to the operator (encoded by

Lactose utilization in an E coli cell

lac

Z Y A

Encodes permeaseEncodes β-galactosidase

A

Encodes transacetylase

Lactose utilization in an E coli cell

•Permease

transports lactose into cell

The utilization of lactose by E.coli: A model system

for gene regulation

Lactose utilization requires two enzymes

• Permease and β-Galactosidase (β-Gal)

In the absence of lactose, both enzymes are present at very

Repression of lac gene expression

In the absence of lactose, repressor binds to the operator and prevents transcription

lac repressor is a negative regulatory element

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Induction of the lac operon in E coli

When lactose is present:

• Inducer binds to the lac

• RNA polymerase binds

to the promoter and

initiates transcription of

the polycistronic lac

mRNA

Inducer: allolactose, IPTG

16

Advantages of using lactose utilization by E coli as

a model for understanding gene regulation

Lac − mutants can be maintained on media with glucose

and so lac genes are not essential for survival

• If both glucose and lactose are present, E coli cells

will use glucose first

Simple assays for lac expression - use of ONPG or X-gal

as substrates for β-gal (color change)

Lactose induces a 1000-fold increase in β-gal activity

Detection and characterization of hundreds of lac − mutants

defective in lactose utilization

lacl− mutants have a mutant repressor that

cannot bind to operator

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In lacI − mutants, lac genes are expressed in the absence and

the presence of inducer (constitutive expression)

lacls mutants have a superrepressor that binds

to operator but cannot bind to the inducer

In lacI S mutants, lac genes are repressed in the absence

and the presence of inducer

lacOc mutants have a mutant operator that cannot

bind the repressor

In lacO c mutants, lac genes are expressed in the absence and the presence of inducer (constitutive expression)

The PaJaMo experiment provided

evidence that lacI encodes a repressor

lacI + lacZ + DNA

transferred into lacI −

of inducer

The lac repressor binds to operator DNA,

W Gilbert et.al., 1966

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DNase footprint establishes the repressor- binding region

How the inducer acts to trigger synthesis

of lac enzymes

Binding of inducer to repressor changes the shape of the

repressor so that it can longer bind to DNA

• When no inducer is present, repressor is able to bind to DNA

Repressor is an allosteric protein – undergoes reversible

changes in conformation when bound to another molecule

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lac repressor has two separate domains

Mutated sequences in

many different lacI −

mutants clustered in the

DNA-binding domain

Mutated sequences in

many different lacI S

many different lacI S

mutants clustered in the

inducer-binding domain

X-ray crystallography

revealed the two

separate domains

Proteins act in trans, DNA sites act in cis

Jacob and Monod used partial diploids carrying different

alleles of lac regulatory elements and structural genes to identify trans-acting and cis-acting elements

Trans-acting elements:

• Can diffuse through the cytoplasm and act at target

DNA sites on any DNA molecule in the cell

Proteins act in trans, DNA sites act in cis

Lacl+ protein acts in trans

Inducible synthesis

Repressor expressed from the plasmid can diffuse through the cytoplasm and bind to the operator on the chromosome

Lacls protein acts in trans

Noninducible synthesis

Superrepressor expressed from the plasmid can diffuse through the cytoplasm

lacOc acts in cis

Constitutive

The lacO C mutation affects expression of genes only on the DNA that it

is located on

The lac operon of E coli is regulated

by both lactose and glucose

When both glucose and lactose are present, only glucose is utilized

Lactose induces lac mRNA expression, but only in the

absence of glucose

• Lactose prevents repressor from binding to lacO

• lac repressor is a negative regulator of lac transcription

lac mRNA expression cannot be induced if glucose is present

• Glucose controls the levels of cAMP

• cAMP binds to cAMP receptor protein (CRP)

Positive regulation by CRP–cAMP

Catabolite repression – overall effect of glucose is to

prevent lac gene expression

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The CAP protein mediates the effects of cAMP on lac

operon expression

How regulatory proteins interact with RNA polymerase

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CRP–cAMP binds as a dimer to a regulatory

region

CRP-binding sites have a two-fold rotational symmetry

CRP protein binds as a dimer

5'TGTGAGTTAGCTCACA 3' 3'ACACTCAATCGAGTGT 5'

• CRP-binding site consists of two recognition

sequences, one for each subunit of the CRP dimer

DNA recognition sequences by

helix-turn-helix (HTH) motif

A protein with an HTH motif has

two α-helical regions

separated by a turn in the

protein

The HTH motif fits into the

major groove of DNA

One of the α-helices recognizes

a specific DNA sequence

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Jacques Monod and Francois Jacob – Pasteur Institute

• Nobel Prize in 1965 (with A Lwoff) for their discoveries

concerning genetic control of enzyme and virus synthesis

• Operon theory - one signal can simultaneously regulate expression of several clustered genes

The operon theory of gene regulation

expression of several clustered genes

• Hypothesized that lac genes are transcribed together as a

single mRNA (polycistronic) from a single promoter

Regulator Protein

Type of Control Bound

to DNA

Not bound to DNA

Negative (lac repressor)

Operon Off

Operon On

Operon Off (CAP protein)

The lac operon of E coli is regulated

by both lactose and glucose At which conditions the lacZ gene is highly

expressed?

AraC acts as both a repressor and an activator

Using the lacZ gene as a reporter of gene

expression

Reporter gene – protein-encoding gene whose expression in the cell is quantifiable by sensitive and reliable techniques

Measuring gene expression

• Fuse coding region of lacZ to cis-acting regulatory regions

from other genes

Identifying sets of genes regulated by the same stimulus

• Create library of cells with promoter-less lacZ inserted by

transposition into random sites in the genome

Controlling gene expression

• Fuse the lac regulatory sequences to the coding region of a

foreign gene

• Inducible expression of the foreign gene controlled by IPTG

lacZ fusion used to perform genetic

studies of the regulatory region of gene X

Conditions that regulate expression of the test regions from

gene X will alter the levels of β-galactosidase

Specific regulatory sites can be identified by constructing

and testing mutations in the test regions of gene X

Histochemical localization of GUS expression under control

of the Atndt1 and Atndt2 promoter in A thaliana.

General Genetics-BIO1053

7 th week

Using lacZ to identify sets of genes regulated by the

same stimulus

Transposition of

promoter-less lacZ

coding region

Library of clones containing

lacZ insertions at random sites

Screen library to identify all the

genes that express lacZ in

Use of fusions to

overproduce a gene

product

Expression of gene X under control

of the lac regulatory system

Expression of human growth

hormone in E coli controlled by

lac control region

Regulation of the tryptophan (trp)

operon in E coli

Structural genes for tryptophan (Trp) biosynthesis are expressed only in the absence of Trp

Two mechanisms for trp operon regulation

• TrpR gene encodes the trp repressor that can bind to the Trp

operator (TrpO)

• When Trp is present, TrpR repressor binds to TrpO

• When Trp is absent, TrpR repressor cannot bind to TrpO

• Attenuation controls termination of transcription in the trp

leader (TrpL)

Tryptophan acts as a corepressor

Evidence that TrpR repressor is not the

only regulator of the trp operon

Constitutive expression of Trp biosynthesis doesn't occur in

TrpR − mutants

If TrpR were the sole regulator, maximal expression of trp genes

would occur in the absence or presence of tryptophan

Second regulatory mechanism is attenuation – control of gene Second regulatory mechanism is attenuation – control of gene expression by premature termination of transcription

Attenuation controls termination of transcription in the trp

leader (TrpL)

• Truncated mRNA - terminates in TrpL, only 140 bases

• Full-length mRNA - continues through TrpL and encodes

all five structural genes

Transcription from the trp promoter produces

two alternative mRNAs

all five structural genes

Alternate stem-loop structures in trpL RNA

Different regions of trpL have complementary base-pairing

• Formation of the 1-2 stem-loop allows formation of the 3-4 stem-loop

• Formation of the 2-3 stem-loop prevents formation of the 3-4 stem-loop

• The 3-4 stem loop is a transcription terminator

When tryptophan is present, transcription terminates

The trpL mRNA is translated and includes two trp codons

Movement of ribosomes through trpL mRNA depends on the

availability of tRNA Trp

• When Trp is present, tRNA Trp is available and rapid ribosome movement allows the formation of 3-4 stem-loop

When tryptophan isn't present,

transcription doesn't terminate in trpL

Sigma factor ( σ ) recognition sequences

Normal, housekeeping sigma factor is σ70

• Active under normal physiological conditions, but is inactivated by heat shock

• Heat shock inducible genes have promoters that are recognized by σ32

recognized by σ

• σ32 is resistant to inactivation by heat shock

Sigma factor ( σ ) recognition sequences

Binding of RNA polymerase to promoter

The promoter sequence of the lac operon is TATGT T at the -10 region, and

TT T ACA at the -35 region.

The consensus sequence of σ 70 which regulates the lac operon is TATAAT at

the -10 region, and TTGACA at the -35 region.

They differ at three positions: two at the -10 region and one at the -35 region.

Experimentally, it has been found that binding of the RNA polymerase to the

lac promoter is relatively weak.

Translational control of another sigma

factor encoded by the rpoS gene

Under normal conditions, rpoS gene is transcribed but rpoS

mRNA is not translated

After stress response, a small RNA (dsrA) binds to rpoS

mRNA and allows translation