4.4-Lambda Early Transcription Overview

Overview

Lecture covers bacteriophage lambda early response and progression toward the lytic cycle, emphasizing transcriptional control, anti-termination, and host factor interactions. Includes course reminders on homework and exams.

Announcements

• Homework due tomorrow by 11:00 p.m.
• Exam next week; focus: bacteriophage lambda, protein chaperones, protein secretion, tmRNAs.
• Grading update: last exam grades expected by Wednesday.

Lambda Infection and Early Transcription

• Lambda binds E. coli via LamB receptor using tail fiber J protein; tail compresses and injects DNA.
• DNA crosses periplasm and enters via PtsM (Enzyme II C/II D), a mannose permease channel.
• Genome circularizes; strong promoters PL, PR, PR′ recruit RNAP with σ70 without activation.
• Excess copies of strong promoters can sequester RNAP and kill cells.

Immediate Early Genes and Termination

• PL makes short transcript encoding N; terminates after N ORF.
• PR makes short transcript encoding CRO; terminates after cro ORF.
• PR′ transcribes a short non-coding transcript initially; later functional.
• Early terminators are Rho-independent; stem-loops trigger NusA-mediated pausing and termination.

N-Dependent Anti-termination

• Nut sites (N utilization sites) flanking PL and PR transcribed into RNA elements with boxA and boxB.
• BoxB stem-loop binds N protein; NusA, NusG, and other Nus proteins assemble an anti-termination complex.
• Complex prevents terminator stem-loop from pausing RNAP; PL and PR transcripts extend to downstream genes.
• Anti-termination occurs on both PL and PR when N levels are sufficient.

CRO Regulation at the Right Operator

• cro is made even without anti-termination; CRO is a transcriptional repressor.
• Operator sites OR1, OR2, OR3 upstream of PR/PRM regulate promoter activities.
• CRO binds OR3 with highest affinity at low CRO; represses PRM (opposite direction promoter).
• As CRO rises, binds OR2 then OR1; OR1 binding represses PR, lowering cro transcription.
• Negative feedback: CRO auto-regulates its concentration, preventing excessive repression of PR.

Extended Transcription from PL: cIII, xis, int, sib

• Anti-terminated PL transcript includes cIII, xis, int, and sib sequence.
• CIII (c3) inhibits E. coli FtsH, an ATP-dependent protease.
• xis encodes excisionase; required to excise prophage during induction from lysogeny.
• int encodes integrase; mediates integration into E. coli chromosome via homologous recombination.
• sib is a regulatory RNA sequence; when co-transcribed with int, sib base-pairs over int to block int translation.
• Integrase production requires specific conditions separating int from sib effect.

Extended Transcription from PR: cII, O, P, Q

• cII encodes a transcriptional activator; targets weak promoters linked to lysogeny.
• cII is a strong substrate for FtsH; rapidly degraded without protection.
• O and P recruit E. coli replication machinery (e.g., DnaA) to lambda ori; essential for phage DNA replication.
• Q encodes a distinct anti-terminator acting later via a different mechanism from N.

DNA Replication Strategies

• Circular lambda genome replicates using host machinery.
• O/P facilitate DnaA loading at ori; initiate bidirectional theta replication.
• After ~5–6 rounds, DnaA becomes limiting; switch to rolling-circle replication.
• Rolling circle displaces a single strand; continuous synthesis yields linear concatemers.
• Concatemers later processed into unit-length genomes for packaging.

cII-Activated Promoters and Lysogeny Bias

• cII activates three promoters: PRE, Pint, and Panti-Q (PQ′).
• These promoters primarily support lysogeny-related transcripts.
• One cII-activated promoter lies within cro but transcribes in the opposite direction.
• cII stability depends on CIII inhibition of FtsH; links gene expression to host proteolysis.

Proteolysis Control: CIII–FtsH–ATP Axis

• FtsH degrades cII efficiently; CIII broadly inhibits FtsH-mediated degradation.
• Western blot evidence: without CIII, cII declines ~90% in 12 min; with CIII, cII maintained.
• In cell lysates, CIII protects multiple substrates (cII, cI-ssrA-tagged, beta-casein) from degradation.
• CIII itself is an FtsH substrate but degrades more slowly; degradation requires ATP.
• Low ATP reduces FtsH activity; CIII persists, allowing cII accumulation.
• High ATP promotes CIII degradation; frees FtsH to degrade cII, lowering cII levels.

Physiological Logic of ATP-Dependent Decision

• Low ATP signals poor host/environmental conditions; favors cII accumulation and lysogeny.
• High ATP indicates robust growth; cII reduced, biasing toward lytic development.

Key Terms & Definitions

• LamB: E. coli outer membrane receptor for lambda.
• PtsM (Enzyme II C/D): Mannose permease channel facilitating DNA entry.
• PL, PR, PR′: Strong sigma-70 promoters initiating early transcription.
• N protein: Anti-terminator binding nut RNA to enable readthrough.
• nut (boxA/boxB): RNA elements recruiting N and Nus proteins for anti-termination.
• NusA/NusG: Host factors in transcription termination/anti-termination.
• CRO: Repressor regulating PRM and PR via OR sites; auto-regulates its level.
• OR1/OR2/OR3: Operator sites controlling PR and PRM.
• PRM: Promoter for maintenance repressor; repressed by CRO at OR3.
• cIII (C3): Inhibitor of FtsH protease; stabilizes cII and other proteins.
• FtsH: ATP-dependent protease degrading cII, cIII, and others.
• xis: Excisionase enabling prophage excision.
• int: Integrase mediating genome integration into host chromosome.
• sib: RNA sequence blocking int translation when co-transcribed.
• cII (C2): Activator of PRE, Pint, Panti-Q; promotes lysogeny.
• O/P: Lambda proteins recruiting DnaA and replication machinery.
• Q: Anti-terminator for late gene expression (distinct mechanism from N).
• Theta replication: Bidirectional replication of circular DNA.
• Rolling-circle replication: Unidirectional fork generating concatemers.

Structured Elements

Action Items / Next Steps

• Review mechanisms of N and Q anti-termination; Q to be covered next lecture.
• Study operator-promoter logic (PR/PRM with CRO) and cII/cIII–FtsH interactions.
• Understand replication shift from theta to rolling circle and roles of O/P.
• Complete homework by tomorrow 11:00 p.m.; prepare for next week’s exam topics.