Regulation of Gene Expression in Bacteriophage (lambda)

 

Bacteriophage

>Virus infecting E.coli

>Temperate phage

>50kb ds linear genome in phage

>circular in host

>cos (cohesive ends) site overhangs of 12 bases at 5' ends

>Ligated using DNA ligase and gyrase

>Upon infection, phage can undergo lysis or lysogeny

>Health of host cell determines fate of lambda

Lambda Life Cycle

Lysis

>If cell is healthy, sufficient energy is available for production of progeny phage and lambda phage will go lytic

>3 phases - immediate early, delayed early and late lysis

>production of new phage particles

>replication of lambda phage genome

>production of viral coat proteins

>assembly of progeny phage

>lysis of host cell

Lysogeny

>If cell is unhealthy, lambda will go lysogenic

>3 phases - immediate early, delayed early and late lysogeny

>viral genome integrates into host chromosome passively

>excision of genome from host chromosome by induction when under stress

>phage DNA - prophage

>cell harboring - lysogen

>no progeny produced

>not disadvantageous for host

>Immunity to superinfection (from same phage)

Map of lambda phage

>50 genes coat proteins

>proteins in DNA replication, recombination and lysis

Regulatory Proteins

>Genes clustered into operons

>3 promoters (PR, PL & PRM)

-PR, PL (rightward & leftward)

Strong, constitutive promoters

-Do not need activator proteins

-PRM (Promoter for repressor maintenance)

Weak – directs transcription when activator binds upstream

 2 genes (cI and cro)

>cI - lambda repressor

repress transcription – binds sites that overlap promoter & excludes RNAP

Activate transcription – similar to CAP in activating lac operon; N terminal domain

>Cro (control for repressor and other things)

activate transcription

>Cro and pcI can bind operator sites OR and OL

>operator sites composed of 3 specific operator regions: OR1,, OR2 (overlaps PR, PL), OR3 (overlaps PRM)

Cooperative Binding of cI

pcI 

>2 subunits forming “dumbbell" with DNA binding end and repressor binding end

>forms a dimer to bind DNA

>C terminal provides dimerization contacts and mediates interactions between dimers (tetramerization) – cooperative binding of repressor

>binding affinity: OR1 >>> OR2 = OR3

-binding to OR1 and OR2 is cooperative

-Without cooperativity, 10-fold higher conc of cI needed to bind OR2

Cro

Binds operator in opposite affinity but not cooperatively

cI and cro bind in different patterns

Lysogeny

>low [pcI], binds to both OR1 /OL1 (represses PR /PL) and OR2 /OL2 (activates PRM lambda cI expression)

positive autoregulator

>high [pcI], pcI also binds to OR3 /OL3 - represses transcription of PRM

negative autoregulator

>PR and PL OFF, PRM ON

Lysis

>Cro binds OR3 overlaps PRM represses cI transcription

>RNAP binds PR – transcription of lytic genes

>PR and PL ON, PRM OFF