REGULATION OF GENE

EXPRESSIONPROKARYOTES

REGULATION OF GENE

EXPRESSIONPROKARYOTES

3 LEVELS OF GENE EXPRESSIONREGULATION

OVERVIEW OFREGULATORY MECHANISMS

CONTROL OF GENE EXPRESSION CONTROL OF GENE EXPRESSION IN

PROKARYOTES Enables bacteria to adjust their metabolism to

environmental change Responses to environmental stimuli

REGULATION OF GENE EXPRESSION REGULATION OF ENZYMATIC ACTIVITY

ENZYME REGULATION DURING METABOLISM CONTROL OF ENZYMATIC ACTIVITY

REGULATION BY FEEDBACK INHIBITION

FEEDBACK INHIBITION ISOLEUCINE

SYNTHETIC PATHWAY

CATABOLIC OPERONSCATABOLIC OPERONS

INDUCIBLE ENZYMESINDUCIBLE ENZYMES

CONTROL OF GENE EXPRESSION CONTROL OF GENE EXPRESSION IN

PROKARYOTES» Enzyme synthesis (Regulation of gene expression)

• At the level of transcription of the genes coding for particular enzymes - control the # of enzyme molecules produced

• Slower to take effect than feedback inhibition, but is more economical for the cell. It prevents unneeded protein synthesis for enzymes, as well as, unneeded pathway product

» Examples illustrating regulation of a metabolic pathway is the tryptophan pathway in E. coli. Mechanisms for gene regulation were first discovered for E. coli

» Current understanding of such regulatory mechanisms at the molecular level is primarily limited to bacterial systems

» Reports on some eukaryotes & viruses*Displacements of Prohead Protease Genes in the Late Operons of Double-Stranded-DNA Bacteriophages".

Journal of Bacteriology. 1 March 2004. Retrieved 30 December 2012.

CONTROL OF GENE EXPRESSION OPERON MODEL-François Jacob and Jaques Monod

(1961)-Regulated genes can be switched on/off depending on cell's metabolic needs Basic Definitions

» Operon = A group of clustered genes that produces a single messenger RNA molecule in transcription and that consists of structural genes and regulating elements

» Structural gene = Gene that codes for a polypeptide• Common in bacteria and phages• Has a single promoter region, so RNA polymerase will

transcribe all structural genes on an all/none basis• Transcription produces a single polycistronic mRNA with

coding sequences for all enzymes in a metabolic pathway • Translation —> separate polypeptides

lac OPERON

CONTROL OF GENE EXPRESSION OPERON MODEL-François Jacob and Jaques Monod

(1961) Basic Definitions

» Polycistronic mRNA = A large mRNA molecule that is a transcript of several genes

• Is translated into separate polypeptides• Contains stop and start codons for the translation

of each polypeptide» Grouping structural genes into operons provides an

advantage b/c:• Expression of all genes can be coordinated. When

a cell needs the product of a metabolic pathway, all the necessary enzymes are synthesized at one time.

• The entire operon can be controlled by a single operator

OPERONoperons.swf

CONTROL OF GENE EXPRESSION IN PROKARYOTESTHE LACTOSE UTILIZATION OPERON

Basic Definitions» Inducible operon» Operator-(between promoter and structural

genes/within promoter)-controls access to RNA polymerase to the structural genes

» Repressor protein-binds to the operator and blocks the attachment of RNA polymerase to the promoter

• Repressor protein encoded by regulatory gene• Corepressor-usually observed in biosynthetic

operons (trp operon)» Structural genes» lac operon-a catabolic operon

• lacI-regulatory gene-encodes repressor protein

CONTROL OF GENE EXPRESSION IN PROKARYOTESTHE LACTOSE UTILIZATION OPERON

Structural Genes» lacZ--galactosidase (lactose Glucose + Galactose)» lacY-permease» lacA-transacetylase

Inducer-allolactose (an isomer of lactose)» Inducer present-operon active-synthesis of enzymes

for metabolism of lactose» Inducer absent-operon inactive-active repressor

binding to the operator prevents access to RNA polymerase

• Basal levels of lactose metabolic enzymes due to unstable interaction between repressor protein and operator

AN EXAMPLE OF NEGATIVE REGULATION

(b) Lactose present, repressor inactive, operon on

(a) Lactose absent, repressor active, operon off

mRNA

Protein

DNA

DNA

mRNA 5

ProteinActiverepressor

RNApolymerase

Regulatorygene

Promoter

Operator

mRNA5

3

Inactiverepressor

Allolactose(inducer)

5

3

NoRNAmade

RNApolymerase

Permease Transacetylase

lac operon

-Galactosidase

lacYlacZ lacAlacI

lacI lacZ

Animation Ch. 8 Operons Induction

lac OPERON

LAC REPRESSOR INTERACTING WITH DNA

(Constitutive synthesis)

OVERVIEW POSITIVE & NEGATIVE CONTROL OF GENE

EXPRESSION

This one

Lac operon: CAP (Catabolic activator protein)

POSITIVE REGULATION OF THE LAC OPERON Positive control of a regulatory system occurs only if an activator

molecule interacts directly with the genome to turn on transcription (lac operon)

lac operon is under dual regulation that includes negative control by repressor protein and positive control by cAMP receptor protein (CAP)

CAP (gen: crp) = An allosteric protein that binds cAMP and activates transcription binding to an operon's promoter region (enhances the promoter's affinity for RNA polymerase)

cAMP-CAP-positive activator of lactose metabolic enzyme synthesis (facilitates RNA pol. binding to the promoter-if glucose is absent

Glu absent: cAMP high Glu present: cAMP low

lac OPERON

lac OPERON POSITIVE CONTROL

E. coli preferentially uses glucose over lactose as a substrate for glycolysis (Higher efficiency)

Therefore, normal expression of the lac operon requires:» Presence of lactose» Absence of glucose (crp:cAMP receptor protein

gene ) When Glu concentration decreases, cAMP increases

lac OPERON POSITIVE CONTROL How is CAP affected

by the absence or presence of glucose? When glucose missing,

cell accumulates cAMP, a nucleotide derived from ATP.

cAMP activates CAP so that it can bind to the lac promoter

When glucose concentration rises, glucose catabolism decreases the cAMP concentration

POSITIVE REGULATION OF THE lac OPERON

[GLUCOSE]

[cAMP] rises

cAMP binds CAP

cAMP-CAP complex binds lac promoter

Efficient transcription of lac operon

[cAMP] becomes scarce

cAMP loses CAP

CRP disengages fromlac promoter

Slow transcription of lac operon

AbsentPresent

lac OPERON

QuickTime™ and aCinepak decompressor

are needed to see this picture.

lac operonhttp://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html

* lac operonhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.html

lac operonoperons_induction.swf

Self quiz link: http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.html

DUAL REGULATION OF lac OPERON IN THIS TYPE OF REGULATION

Negative control by repressor determines whether or not the operon will transcribe the structural genes

Positive control by CRP determines the rate of transcription (slow vs. efficient)

E. coli economizes on RNA/protein synthesis with the help of these negative and positive controls

CRP is an activator of several different operons that program catabolic pathways

Glucose's presence deactivates CRP. This, slows the synthesis of those enzymes a cell needs to use catabolites other than glucose

E. coli preferentially uses glucose as its primary carbon and energy source, and the enzymes for glucose catabolism are coded for by unregulated genes that are continuously transcribed (constitutive)

DUAL REGULATION OF THE lac OPERON IN THIS TYPE OF REGULATION

Therefore, when glucose is present, CRP does not work and the cell's systems for using secondary energy sources are inactive

When glucose is absent, the cell metabolizes alternate energy sources» The cAMP level rises, CRP is activated and

transcription begins of operons that program the use of alternate energy sources (e.g., lactose)

» Which operon is actually transcribed depends upon nutrient availability

• Example: If lactose is present, the lac operon will be switched on as allolactose inactivates the repressor

ANABOLIC OPERONSANABOLIC OPERONS

REPRESSIBLE ENZYMESREPRESSIBLE ENZYMES

REPRESSIBLE OPERONS REPRESSIBLE ENZYMES

Their synthesis is inhibited by the specific metabolite»trp operon

• trp present-operon inactive-trp is the corepressor• trp absent-operon active

BACTERIA ARE REMARKABLE IN THEIR ABILITY TO ADAPT TO A VARIETY OF ENVIRONMENTSS BY THEIR ELABORATE CONTINGENCY OF MECHANISMS TO CONTROL ENZYME SYNTHESIS AND HENCE METABOLIC PATHWAYS

Polypeptide subunits that make upenzymes for tryptophan synthesis

(b) Tryptophan present, repressor active, operon off

Tryptophan(corepressor)

(a) Tryptophan absent, repressor inactive, operon on

No RNA made

Activerepressor

mRNA

Protein

DNA

DNA

mRNA 5

Protein Inactiverepressor

RNApolymerase

Regulatorygene

Promoter Promoter

trp operon

Genes of operon

Operator

Stop codonStart codon

mRNA

trpA

5

3

trpR trpE trpD trpC trpB

ABCDE

trp OPERON

QuickTime™ and aCinepak decompressor

are needed to see this picture.

Animation Ch. 8 Operons repression

trp operon http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html

Additional regulation mechanism: Attenuation of trp operon:http://www.youtube.com/watch?v=8aAYtMa3GFU

REPRESSIBLE VS. INDUCIBLE OPERONS REPRESSIBLE (trp)

Their genes are switched on until metabolite activates the repressor

They function in anabolic pathways

Pathway’s end product switches off its own production by repressing enzyme synthesis

ANABOLIC

INDUCIBLE (Lac) Their genes are

switched off until a specific metabolite inactivates the repressor

They function in catabolic pathways

Enzyme synthesis is switched on by the nutrient the pathway uses

CATABOLIC