MICB404, Spring 2008 Lecture #25 Operons Microbial Genetics MICB404, Spring 2008 Lecture #25 Operons

Announcements Today’s lecture 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

lac operon (lactose, allolactose, etc.) LacI

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)

lac operon genetics Complementation and cis/trans tests F’ plasmid bearing lac mutations Conjugate with recipient bearing chromosomal lac mutations Score phenotype of transconjugant

lac operon genetics lacI- lacZ+ lacY+ lac A+ Operon 1 Operon 2 Inducer present Inducer absent Recessive or dominant? cis or trans?

lac operon genetics lacI+ lacOc lacZ+ lacY+ lac A+ Operon 1 Operon 2 lacI+ lacO+ lacZ+ lacY+ lac A+ Repressor binds only partially to Oc Inducer present Inducer absent

lac operon genetics lacI+ lacOc lacZ+ lacY+ lac A+ Operon 1 Operon 2 lacI+ lacO+ lacZ- lacY+ lac A+ Repressor binds only partially to Oc Inducer present Inducer absent

lac operon genetics lacI+ lacOc lacZ- lacY+ lac A+ Operon 1 Operon 2 lacI+ lacO+ lacZ+ lacY+ lac A+ Repressor binds only partially to Oc Inducer present Inducer absent

lac operon genetics lacI+ lacOc lacZ- lacY+ lac A+ Operon 1 Operon 2 lacI+ lacO+ lacZ+ lacY+ lac A+ 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. cis or trans? Recessive or dominant?

Summary of lac mutations Expression Relation to wildtype Action I+ Inducible trans I- Constitutive Recessive IS Non- Dominant ID OC (partially) 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 Diauxic shift [lactose] [glucose]

Catabolite repression high [glucose]  low [cAMP]i As glucose is consumed, intracellular [cAMP] increases ATP cAMP adenylate cyclase (cya)

Catabolite repression 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 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] low CAP-cAMP doesn’t bind to lacP and transcription not activated

Catabolite repression [cAMP] high CAP-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

trp operon regulation 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

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 cAMP CAP Pentose Phosphate Pathway

ara operon AraC binds arabinose P1 state: no arabinose bound P2 state: arabinose bound, and functions as transcriptional activator

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 arabinose 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

ara operon AraC autoregulation at low arabinose, AraC binds araO2 and araI1 and prevents araC transcription at high [AraC], it binds to araO1

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

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