Microbial Genetics MICB404, Spring 2008 Lecture #25 Operons

Microbial Genetics

MICB404, Spring 2008Lecture #25

Operons

• Announcements– Spring

break!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

• Today’s lecture– lac operon

Operons• “functionally integrated genetic units for

control of gene expression. Consist of structural genes, and of adjacent sites (promoter and operator) that control transcription of the structural genes”– structural genes involved in shared metabolic

pathways, protein complexes, or cellular functions– coordinated regulation of gene expression

• transcriptional initiation (repression, activation)• transcriptional termination• post-transcriptional regulation

– mRNA stability, translation– protein stability, activity

LacI

lac operon

(lactose, allolactose, etc.)

Complementation and cis/trans tests

• Dominant vs recessive mutations- recessive: does not exert phenotype if wildtype allele is present- dominant: exerts phenotype even if wildtype allele is present

• cis vs. trans acting mutations- cis: typically on a non-coding regulatory DNA (affects only that DNA and can’t be complimented) - trans: usually affects a diffusible gene product (can be complimented)

• Complementation and cis/trans tests– F’ plasmid bearing lac mutations– Conjugate with recipient bearing

chromosomal lac mutations• Score phenotype of transconjugant

lac operon genetics

lac operon genetics

lacI- lacZ+ lacY+ lac A+

lacI+ lacZ+ lacY+ lac A+

Recessive or dominant?

cis or trans?

Inducer present

Inducer absent

Operon 1

Operon 2

lac operon genetics

lacI+ lacOc lacZ+ lacY+ lac A+

lacI+ lacO+ lacZ+ lacY+ lac A+

Inducer present

Inducer absent

Operon 1

Operon 2

Repressor binds only partially to Oc

lac operon genetics

lacI+ lacOc lacZ+ lacY+ lac A+

lacI+ lacO+ lacZ- lacY+ lac A+

Inducer present

Inducer absent

Operon 1

Operon 2

Repressor binds only partially to Oc

lac operon genetics

lacI+ lacOc lacZ- lacY+ lac A+

lacI+ lacO+ lacZ+ lacY+ lac A+

Inducer present

Inducer absent

Operon 1

Operon 2

Repressor binds only partially to Oc

lac operon genetics

lacI+ lacOc lacZ- lacY+ lac A+

lacI+ lacO+ lacZ+ lacY+ lac A+

cis or trans?Recessive or dominant?

Operon 1

Operon 2

As long as there is a functional lacZ gene adjacent to a Oc operator, β-galactosidase expression will be observed even if a wildtype operator is present in the same cell.

Summary of lac mutationsMutation Expressio

nRelation

to wildtype

Action

I+ Inducible trans

I- Constitutive

Recessive trans

IS Non-Inducible

Dominant trans

ID Constitutive

Dominant trans

OC Constitutive(partially)

Dominant cis

lac operon genetics• Mutation in lacZ and lacY more

common than in lacO or lacP– target size

• lacO is divided into 3 operator sites– LacI as tetramer

binds to them can bind to them indi-vidually or in pairs

– bent promoter unable to recruit RNA polymerase

Catabolite repression

• Glucose is carbon source of choice for E. coli– Diauxic growth: glucose is completely

consumed before lactose consumption begins

Diauxic growth

[glucose]

[lactose]

Diauxic shift

• high [glucose] low [cAMP]i

• As glucose is consumed, intracellular [cAMP] increases

Catabolite repression

ATP cAMP

adenylate cyclase (cya)

• high [glucose] low [cAMP]I

– how?

• Glucose uptake via PhosphoTransferase System (PTS)– One component of PTS (IIAGlc) is

dephosphorylated upon glucose uptake

– IIAGlc-PO4 activates adenylate cyclase

– If IIAGlc is participating in glucose transport, it is not able the activate adenylate cyclase

Catabolite repression

• cAMP binds with CAP protein– “catabolite activator protein”– also called CRP

• CAP-cAMP binds to lac promoter– increases efficiency of RNA polymerase

transcriptional initiation

Catabolite repression

• High glucose low cAMP low transcription

• High lactose LacI released from operator

• Low glucose + high lactose expression of lac operon

Catabolite repression

Sugars in growth medium β-galactosidase activity (relative)

glucose 1

glucose + lactose 50

lactose 2500

Catabolite repression

• Also acts on mal, gal, ara operons– and others– Global Regulatory Mechanism

Inducer exclusion

• Glucose also prevents uptake of lactose from medium– Component of PTS binds to LacY and

inhibits lactose uptake– By excluding the inducer from the cell,

expression of the operon is further repressed

Catabolite repression

[cAMP] lowCAP-cAMP doesn’t bind to lacP and transcription not activated

Catabolite repression

[cAMP] highCAP-cAMP binds to lacP and transcription potentially activated

If lactose is present, repression is removed and lac operon expressed

CAP protein

• Contacts RNA polymerase• Together, these result in activation of

lac operon transcription

trp operon

• Tryptophan is required for protein synthesis and hence growth– E. coli can use exogenous Trp– When unavailable, synthesis is required

• trp operon• Five structural genes encoding three

enzymes required to convert chorismic acid into tryptophan

trp operon

• Anthranilate synthetase – trpE, trpD• N-(5’-phosphoribosyl)-anthrnilate isomerase/Indole-3-glycerol

phosphate synthase – trpC• Trytophan synthase – trpB, trpA

•When trp repressor bound to operator, transcription does not occur

trp operon regulation

• Expression of trpEDCBA is reduced by the addition of tryptophan in trpR mutants

• Lead to discovery of a 2nd level of trytophan control

- attenuation

trp operon regulation

• Attenuation

- tryptophanyl-tRNATrp tRNATrp charged with tryptophan

- trpL gene, a non-coding leader sequence at the 5’ end

trp operon regulation

string of T’s (in DNA)

stem-loop

• Base-pairing– Stem-loops 1-2 and 3-4 (terminator), or– Stem-loop 2-3 (anti-terminator)

trp operon regulation

• Translation of leader peptide deter-mines which stem-loops form – Transcription and translation are

coupled– RNA:RNA base pairing is eliminated in

region in contact with ribosome– Formation of stem-loops 1-2 and 3-4 is

mutually exclusive of formation of stem-loop 2-3

trp operon regulation

– Trp codons make translation sensitive to [Trp]• At low [Trp], translation

stalls at Trp codons• Stem-loop 2-3 forms

• Transcription does not terminate

• trp enzymes expressed

trp operon regulation

• At high [Trp], leader peptide translated completely– Ribosome contacts segment 2,

preventing formation of stem-loop 2-3– Stem-loop 3-4, transcriptional

terminator, forms– trp enzyme mRNA

is not transcribed

trp operon regulation

• Fine control• At intermediate [Trp] abundance only

some ribosomes will stall resulting in low-level expression of Trp biosynthetic enzymes– Interplay between TrpR repressor

activity and attenuation

• Attenuation mechanisms common for amino acid biosynthetic operons– His, Thr, Leu, Phe, Ile-Val

trp operon regulation

ara operon

• Regulated by araC gene product• araO1, AraC binds and represses its own transcription. With arabinose, AraC bound at this site activates PBAD.

• araO2, AraC bound at this site can simultaneously bind to the araI site to repress PBAD.• araI, is also an inducer site. In the presence of arabinose, AraC bound at this site helps activate PBAD. • CAP binding, when arabinose is present, it promotes the rearrangement of AraC (repression of PBAD to activation of PBAD)

ara operon• Arabinose: 5-C sugar

PentosePhosphatePathway

CAP

cAMP

• AraC binds arabinose– P1 state: no arabinose bound– P2 state: arabinose bound, and

functions as transcriptional activator

ara operon

ara operon

• In absence of arabinose, AraC (P1 state) binds to araI1 and araO2

• DNA loops out and RNA polymerase cannot access pBAD

ara operon

• In presence of arabinose, AraC (P2 state) is induced to bind araI and 01

• Operon’s pBAD promoter becomes accessible

•CAP-cAMP helps AraC assemble on activator sites and activate transcription from pBAD

arabinose

ara operon• AraC autoregulation

- at low arabinose, AraC binds araO2 and araI1 and prevents araC transcription

- at high [AraC], it binds to araO1

and prevents araC transcription

ara operon• Regulation

– Autoregulation of araC– cAMP/CAP and catabolite repression – Activation of AraC by conformational

change upon arabinose binding– Alteration of AraC activity by adjacent

binding of cAMP-CAP

Spring break

• Study hard!