Mbb 331 Lec 14

Lysis: phage genes transcribed in set order, phage genome is replicated and packaged, the host is lysed

Lysogeny: phage inserted into host genome via site-specific recombination;the prophage replicated with bacterial genome

• LYSIS happens if late genesare expressed

• LYSOGENY happens ifλ repressor synthesis isestablished

• (1) early infection: induction of genesfor DNA replication

• (2) late infection: DNA replication andsynthesis of phage particle proteins

cascade means that the gene expressed at one stagerequired for expression of genes innext stage

works at nut site, permits transcription of delayed early genes

suppresses the action of terminator sequences

allows RNA polymerase to ignore the terminators and continue to transcribe the delayed early genes

Transcription stops at therho-dependent terminators (t)

controlled by P(L)

repressess transcription

Blocks transcription of the λ repressor gene, cI

cI gene encodes λ repressor, P(R)

O and P code for proteins necessary for phage DNAreplication

another antiterminator, Q allows transcription of late genes downstream of the late promoter PR ́

leads to LYSIS & reinfection

N and cro are early, and they are transcribe opposite direction of the promoters P(L) and P(R)

RNAP stops at rho-dependent terminators t(L1) and t(R1) in the absence of N gene product

in the absence of N, RNA transcripts terminates at T(L1) and makes only N mRNA product

In the presence of N, N binds to the transcript of the N utilization site (utilizing the antiterminator activity) and interacts with host proteins to allow transcription to proceed through the terminators into the delayed early genes

Host protein: NusA, NusB, NusG and ribosomal S10 protein

1. N binds NusA, which interacts directly with RNApolymerase

2. N binds box B of the nutsite

3. Nus B binds to box A of the nut site region

(a) weak, nonprocessive complex. NusA binds to RNA polymerase,and N binds to both NusA and box B of the nut site region of thetranscript, creating a loop in the growing RNA. This complex isrelatively weak and can cause anti-termination only at terminatorsnear the nut site

Strong, processive complex is formed when NusA protein tethersN and box B to the polymerase, as in panel (a) but with additionalbinding of S10 and NusB bound to box A at the nut site region of thetranscript. This provides a stronger link between the RNApolymerase and the transcript, strengthening the complex. NusGalso contributes to the strength of the complex. This complex isprocessive and can cause anti-termination thousands of base pairsdownstream in vivo

In the absense of N, NusA facilitates hairpin formation to stimulate termination by trapping the elongation complex

N interferes with terminator hairpin formation, transcription of delayed early genes proceeds

clear not wild-type turbid plaques, turbid plaques mean established lysogeny, clear plaques mean that all host cells are lysed by phage infection,

So, cI and either of cII or cIII mutants cannot establish lysogeny

cll and clll, lysogeny ensues ifsynthesis of a generegulator, the λ repressoroccurs (encoded by the cIgene)

lysogeny ensues if synthesis of a generegulator, the λ repressor occurs (encoded by the cI gene), Delayed early genes are needed for lysogeny

cl gene product: the phage repressor

PRE: Promoter for Repressor Establishment

PRM: Promoter for Repressor Maintenance

• cII protein helps RNA polymerase bind to PRE

• cII also stimulates transcription of the int gene (from PL) resulting in λ DNA integrating into the host genome

continuous supply of repressor

• Repressor Turns off further transcription from PR() and P(L)

• λ repressor binds as a dimer to operators O(R) and O(L)

• Cooperative binding between OR1 and OR2

• λ repressor stimulates own synthesis by activating PRM

• Repressor dimer bound to OR2 contacts RNAP and strengthens its interaction with PRM which stimulates transcription

• λ repressor acts as an activator and binds to OR2(acts as an activator site)

• λ repressor binds to OR3 with the lowest affinity

Compensatory intergenic suppressor mutations in RNAP can suppress repressor mutants defective in PRM transcriptional activation and confirms that RNAP and CI repressor directly interact at P(RM)

High levels of repressor can repress transcription from P(RM) due to COOPERATIVE BINDING (DNA looping) of repressor to sites 1, 2 then 3!

“Tetramerization” denotes the region where two dimers interact when binding cooperatively to adjacent sites on DNA.

• Repressor bound at OR2 contacts RNAP at P(RM), activating expression of the cI (repressor) gene.

OR3 lies within PRM; Cro bound there represses transcription of cI.

• 27 kDa repressor subunit = 2domains joined by connector

• N-terminal domain = DNA binding(operator) has 5 α-helices, two of which are used to bind DNA andconstitute the helix-turn-helixmotif

• C-terminal domain = dimerization with another repressor’s CTD

• Lytic cycle initiated by cleavage ofdimeric repressors in connectorregion

• Cl and Cro are both helix-turn-helix repressors (transcription factors)

• in both CI and Cro, 2 helix-3 amino acids are conserved (neighbouring Gln and Ser) that bond to specific base pairs in DNA

• helix-2 mediates H-bonding with phosphate backbone but does not controlspecificity of target recognition and undergoes ionic interactions withphosphate backbone due to fact that its amino end, which has a net positivecharge, is pointing directly towards negatively charged DNA backbone

OR2 overlaps the -35 regions of PR by 3 bp, and that of P(RM) by 2 bp. This difference is enough for PR to be repressed and PRM activatedby repressor bound at OR2.

In lysogeny, four repressor(CI) monomers are bound at each operator, OR and OL. This shuts down transcription from PR and PL promoters by preventing RNAP binding.

In lysis, no repressors are bound at OR and OL. This opens up PR and PL promoter for RNAP to transcribe delayed early genes.

Decision to infect lytically or lysogenically relies on who wins first: cI or cro

If CI establishes control then lysogeny

If Cro establishes control then lysis

If CI establishes control then lysogeny,

• Repressor bound to OR1 andOR2 turns off transcription from PR • Repressor bound at OR2 contacts RNAP at PRM,activating expression of the cI

lysis

• PRM is blocked

• Cro uses same operator as CI (λrepressor) but with reverse affinity:Cro affinity OR3 > OR2 > OR1

• Cro binds OR3 first and therebyblocks RNAP binding at PRM - notranscription of λ repressor

• All routes for synthesis of λrepressor are blocked and lytic cycleassured

Repressor bound to OR1 and OR2 turns off transcription from PR. Repressor bound at OR2 contacts RNAP at PRM, activating expression of the CI (repressor) gene(autogenous circuit) . OR3 lies within PRM: Cro bound there represses transcriptionof cI.

yes, basically rich medium = lytic, starvation = lysogeny, as[CII] increases, lysogeny increases

• CII sensitive to cellular proteases but are protected from these by CIII

•[Cellularproteases] that degrade CII are higher whencells grown in rich medium, lower during starvation

CII degradation and lytic cycle likely when cells grown in rich medium

• UV uncovers protease activity in the repressor itself

• DNA damage = SOS response

• RecA becomes a co-protease that cleaves the λ repressor

• This is a protective strategy

• Repressor gone = cro transcribed

RNAP stops at rho-dependent terminators tL1, and tR1

qut site and RNAP, Controls transcription of lategenes from PR ́

The products of the delayed early genes cII and cIII alsoparticipate in this process: CII, by directly stimulatingpolymerase to binding to PRE; CIII, by slowing degradation ofCII.