microbial genetics micb404, spring 2008 lecture #25 operons
TRANSCRIPT
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!