Lecture Notes

Week 3 Notes: Transcriptional regulation in bacteriophage

Bacteriophage introduction
 Bacteriophage: bacterial viruses
 3 major morphological classes:
o Icosahedral Tailless: ΦX174
o Icosahedral Tailed: T4, T7, λ
o Filamentous: M13

T-even phages inject their DNA into bacterial cells

Life cycle of phages
 Lytic cycle: DNA replication and lysis of host cell to release progeny phage
 Lysogenic cycle: DNA insertion into a specific site in the bacterial chromosome, latency as a prophage
o Prophage can be induced to excise and enter the lytic cycle

LYTIC DEVELOPMENT
Is divided into two periods
 Early period (before replication) and late period (post replication)
 A phage infection generates a pool of progeny phage genomes that replicate and recombine
o Phage gene function is to ensure preferential replication of phage DNA
 Lytic development accomplished by expression of genes in a certain order

Is controlled by a cascade
 Cascade: sequence of events, each stimulated by previous one
o Transcriptional regulation divided into stages
 At each stage an expressed gene encodes a regulator needed to express gene of next stage
 Early genes: transcribed by host RNA pol following infection
o Include or comprise regulators required for expression of middle genes
 Middle genes: includes regulators to transcribe late genes

 Cascade controlled by two types of regulatory events:
o Regulator proteins promote initiation at new phage promoters or cause host polymerase to read
through terminators (antitermination)
 Control at initiation:
 Replace host enzyme sigma factor w/another factor that redirects specificity to
phage initiation
 Synthesis of new phage RNA pol
 New sets of genes are distinguished by different promoters from those originally
recognised by the host RNA pol

 Control at termination:
 Depends on arrangement of genes: early genes lie adjacent to next set of genes to be
expressed, but are separated by terminator sites
 If termination is prevented, the polymerase reads through into genes on other side
 Same promoters continue to be recognised
Phage lambda
 Lambda can replicate through a lytic cycle or lysogenic cycle
 Lambda genes are clustered according to function
 Cos elements allow circularisation after infection of host

Lambda regulatory region
 PL and PR promoters lie either side of cI gene
o Associated w/each promoter is an operator OL and OR at which repressors bind to prevent RNA pol
from initiating transcription
o Sequence of each operator overlaps w/promoter it controls

, Week 3 Notes: Transcriptional regulation in bacteriophage

 This provides a pressure point at which entry to the cycle can be controlled

 PRM: promoter required for transcription of cI gene (for repressor maintainance)
 PRE : promoter that influences transcription of cI gene (for repressor establishment)

Lytic cycle depends on antitermination by pN
 Lambda has 2 immediate early genes, N and cro, which are transcribed by host RNA pol from form PL and PR
 cro: transcriptional repressor that prevents expression of cI gene
 N gene: encodes antitermination factor which acts at nut sites causing RNA pol to continue transcription past
the ends of the two immediate early genes (overrides termination sequences tL and tR) and transcription of
delayed early genes
 pQ is the product of delayed early gene and is another antiterminator that allows RNA pol to transcribe the
late genes (genes needed for phage assembly)

Overview of lytic infection
 Transcription from PR and PL: N and Cro are transcribed and translated
 Antitermination by N at tL and tR: transcription of delayed early genes, cII, cIII and Q
 Cro (repressor): binds OR- shuts off PR and PRM (cI gene off) and binds OL- shuts off PL.
o All early genes switched off
 Antitermination by Q: activation of PR and transcription of late genes (inc head & tail genes of new phage)

Lambda immediate early and delayed early genes are needed for lytic and lysogenic cycle
 Transcriptional circuit for lytic cycle is interlocked w/circuit for establishing lysogeny
 Both pathways start the same: they require expression of N and cro
 Lysogeny requires delayed early genes cII-cIII and the 2 life cycle pathways diverge
o Critical gene in maintaining lysogeny is the lambda repressor: cI

LYSOGENIC CYCLE
Lambda repressor (LR)
 Maintains lysogeny
 Encoded by cI gene
 Acts at OL and OR operators to block transcription of immediate early genes, thus preventing lytic cycle
from proceeding
o Binding of LR to OR stimulates transcription of cI, its own gene from PRM

 Lysogeny is stable b/c the control circuit ensures that there is continued expression of cI as long as there is
adequate level of LR

 Uses Helix-Turn-Helix motif to bind DNA
o LR binds DNA as a dimer
 Monomeric LR dimersie through their C-terminal domains
o LR N-terminal domain= DNA binding domain, contains 5 aLRha-helices: helices 2 & 3 bind DNA
 DNA binding site is a partially palindromic sequence of 17bp

 LR dimers bind cooperatively to operator
o LR binding to one operator increases affinity for binding a second repressor dimer to the adjacent
operator
 Affinity is 10x greater for OL1 and OR1 than other operators, so they are bound first
o Cooperativity allows LR to bind OL2/OR2 sites at lower concentrations

 LR maintains an autoregulatory circuit
o LR bound at OR2 contacts RNA pol and stabilises/promotes binding to P RM, which promotes
transcription of LR
 This is the basis of autoregulatory control of repressor maintenance