Lac operon and lambda switch Flashcards
(10 cards)
Lac operon overview
System establishes +ve feedback loop w/permease. Molecular principles: cooperativity, regulated recruitment and memory. Operon= simple bistable switch dependent on Lactose concentration.
Beta Galactosidase encoded in Lac operon, cleaves lactose-> beta galactosidase when glucose absent and lactose present. Activity easy to assay, can look for mutants to probe lac operon function. Genetic analysis by complementation, cis/trans, dominance etc-> hypotheses on lac operon organisation.
Lac operon=A, I (trans), Z, Y encode proteins. P, O non-protein coding (regulatory, overlap, cis). Order= IPOZYA.
I encodes a repressor binding O, block RNAP progress, inhibit expression. If allolactose (lactose derivative) /IPTG (effector) binds the repressor protein I, changes its conformation, it can’t bind O, and Z, Y and A are transcribed.
Z= beta galactosidase, Y=permease, A=transacetylase. P=RNAP binding site.
Proof for lac operon through genetic analysis (5 points)
1) Ability to grow on lactose induced by lactose presence
2) Ability to grow on lactose assoc. w/ changes in gene exp (requires protein synthesis), not utilization of proteins pre-existing lactose presence.
3) Most mutations ID genes encoding enzymes (structural genes): Z, Y and A. (found by complementation). Z, Y+ A mutations imply linear organisation (operon)- Some upstream mutations affect downstream genes- “polarity”, suggest collinearity (though most mutations only affect gene they’re in). Molecular studies-> transcribed as 1 mRNA+ translated one after another. All Z/Y/A mutations recessive.
4) Some mutation ID proteins regulating when beta-galactosidase made (I=repressor). I mutants affect ZYA exp. Mutations that lead to beta-galactosidase exp are recessive. Loss of function mutations in I(i-)-> coordinated exp of ZYA (recessive). Other mutations, e.g., iS, system not inducible by lactose- these mutations are dominant- repressor I can’t interact inducer lactose or can’t unbind DNA. For both of these mutants, if I is provided from another piece of DNA, system would act as wt.
5) Some mutations (e.g., o,p) affect regulation (don’t map to protein). o mutants rare, affect I activity and ZYA exp. In OC(constitutive) mutants (cis dominant, can’t be complemented in trans like the I mutants above), repressor can’t bind operator, -> constitutive ZYA exp (similar to i-). OS(super-repressor) mutants (cis dominant, like OC)- repressor can’t unbind, even with IPTG, ZYA can’t be expressed (similar to iS). So
p mutants v rare. Downstream of O, suggests mutations in O act by regulating RNAP activity.
lac: CAP and specificity of transcriptional regulation
CAP: catabolite activating protein, dimer. Mutations have low ZYA expression even when lactose present and in i- double mutants. Low glucose-> cAMP rises, binds CAP (makes it easier to form dimers+ bind DNA)-> CAP binds near P, helps RNAP attach+ promotes its stability, increasing transcriptional initiation rate. Lactose present-> I inactive-> transcription (-> integration of glucose and lactose dependence).
Specificity of transcriptional regulation: I recognition helix can interact ~5bp, not enough to make seq unique- in E-coli genome, need to define minimum 11bp for specificity. Achieve specificity by duplication of 5bp site-> 10bp, and repressor binds as dimer- illustrate general principle of palindromic seq or direct repeats
lac cooperativity
Cooperativity: Given the Kd of repressor/DNA interaction, repression, in principle, determined by # molecules of I. On average, 20 monomers/5 tetramer molecules of repressor per cell. Binding cooperative- 2 molecules help hold each other on DNA for longer, increase interaction stability. Regulated recruitment- helps using molecules for a regulatory event, contributing to event specificity and system sensitivity. In addition to O1 (original operator), O2+O3 on either side, with lower affinity for repressor- repressor binds these too. These sites (far from original) held in place through DNA looping and I tetramers. O2 and O3 seqs play role in repression by increasing cooperativity, and looping fixes DNA and repression. I tends to form tetramers in vitro, so if 5 molecules per cell, about 1 functional molecule of repressor per cell.
Lac physiological memory, feedback circuit
Physiological memory: lactose needs permease to get into cell (encoded by Y). Lactose absent-> Y repressed by I. However, repressor binds and unbinds (1 functional molecule per cell)-> small leakage. Few permease molecules made, bring in some lactose (“sampling” env-system poised for activity). When both glucose eand lactose present, some lactose in cell, Lac operon leaks some mRNA.
Feedback/circuit: when intermediate levels of inducer applied to system, some cells fully induced and some not at all- on/off switch depending on repressor state at the time of exposure. When induced cells selected and placed in intermediate lactose again, remain induced, but uninduced cells still have ability to decide between on/off upon re-exposure. Because once Y exp at certain level-> =ve feedback loop, maintains system active (create memory of input). NB: see a bimodal distribution in beta-galactosidase activity- so distribution>informative than average in this biological case.
lambda: gate type and phenotypes
This e.g., is a NOT gate: if A, then lysis; if B (NOT A), lysogeny. Lysogeny=status quo. Lysis= default associated with a genetic programme. A high level ctrl region determines a seq of events, with a programme following from it. Genetic programme=seq of transcriptional and translational events that change the state of the cell/generate a new state, can lead to other programmes.
Lambda bacteriophage has to decide to kill or not kill host E-coli. Mutants elucidate mechanisms.
2 phenotypes: Infected E-coli lawn exhibits round, clear plaques (O, lytic) where lambda killed bacterium- these plaques never give lysogens when infecting other bacteria and turbid plaques (ø) where lambda only kills some E-coli and growth with the other bacteria, forming lysogens where virus is dormant- bacteria with lysogenic phenotype are immune to further infection. UV treatment of lysogen induces lysis.
lambda: mutant phenotypes and conclusions drawn
1) cI= lysis repressor. If mutated, lambda always lyses-> clear plaques only
2) cII+cIII= establish lysogeny, not required for maintenance so mutants can produce turbid plaques.
3) Can isolate lysogens from ts mutants of cI/II/III at permissive temp (30). cI lyses at 40, but cII/III continue as lysogens- suggests cII/III products needed for initiation/establishment but not maintenance of lysogeny. cI required for both.
4) Cro represses lysogeny, mutants should yield lysogens- but a phage that always produces a lysogen can’t produce clear plaque, so cI and cro have a regulatory relationship.
5) Lambda vir may map to ctrl region in repression, mutants can’t bind repressor. Mutations are cis.
6) Immunity is due to high levels of repressor in host-> supress lytic programme in other phages. DNA of new phage taken over by repressor of resident phage, blocking lysis.
7) UV targets repressor/ break it down.
Cro and the system controlling the lysis/lysogeny decision under normal conditions and lambda vir
Control region and programme on DNA. Programme controlled by proteins. Decision dependent on ctrl region/protein interactions. The operative system, OR region (operator) is mutated in vir- mutants- subdivided into linked sections 1,2,3, with 1 and 3 extremes overlapping promoter for right (PR) and promoter for repressor maintenance (PRM) respectively, and the 3 regions having different and reversed affinities for cro and cI. Proteins cro, cI/II/III emerge as complementation groups.
Cro= simple globular DNA-binding domain. cI= 2 globules linked by hinge, 1 binding DNA (duplicated binding sites) and the other binding another cI-> dimers. Cro and cI both (compete to) bind OR. Competition based on affinities, driven by protein concentrations. OR protein occupancy drives decision to lyse or not.
cI binds OR1 1st, preventing RNAP/PR binding sterically, inhibiting cro exp. Helps RNAP bind PRM, promote cI exp. And phage integration in bacterial chromosome. OR2 also binds well+ cooperatively (binds esp well when OR1 bound). OR3 binds very poorly.
Cro acts in exactly the opposite way (with cooperative binding O3, then 2, then 1). Exp-> lysis.Normal conditions: 90% cells have cI bound, 10% cells all 3 operators occupied-> determinants of outcome= relative concentrations and affinities of cro and cI.
Lambda vir= can be understood as cis dominant mutation (mutants can’t enter lysogeny probably because mutants in operator won’t allow repressor in/favour cro binding-> lytic gene exp, killing host).
3 phases of lambda
Classical genetics and molecular biology approaches establish 3 phases, decision being made between early and late distinguished by mutants decision.
Very early: phage injects DNA, RNAP binds naked DNA of promoters PR and PL (highest affinity)-> cro and N (from PL, downstream of PRM) transcribed. N enables function of RNAP at energetically difficult DNA regions, extending transcription from sites close to PR/L to downstream genes that set up stage for decision making.
Early: from PR, extend exp to cII and genes for lysis. From PL, lead to exp of cIII and genes for integration. cII activates transcription, cIII protects cII from proteolysis (proteases reflect metabolic state of cell and therefore if lambda will replicate or integrate). cII-> cI exp from PRE(promoter for repressor establishment)-> cI starts transcription PRM-> cro and cI competition.
Late: pathways bifurcate. If cII makes enough cI, establish lysogen path. Otherwise cro wins-> lysis.
This is a bistable switch (choice between 2 +ve feedback loops). Key point is if enough cI can be made before cro levels high enough to prevent further exp, which is dependent on cII stability, dependent on protease activity from cell. No possibility of long term coexistence of both pathways.
Rich medium-> activate proteases-> lysis and vice versa.
lac vs lambda comparison
Compare with lac operon
* Both have cis acting operator for DNA binding proteins controlling RNAP.
* Cooperativity of repressor (I) and CAP/ cI and cro. Dimers help tether proteins to DNA
* Regulated recruitment of RNAP (cI, CAP)
* Chief regulatory proteins cI and I act as tetramers-> DNA looping, stabilisation-> energetically favourable structures. +ve feedback loops based on environmental factors-> state stability
* Lambda-> programme activated; Lac-> enzyme made.
* Lambda has 2 bistable regulatory systems- common in bio because robust, mediates choices between states.
