bacteriophage lambda

bacteriophage

• virus that infects bacteria

• phage - virus, uses host’s molecular machinery to reproduce themselves

• different proteins are needed at different stages of replication - different genes expressed at different times

lytic phages

always ready to kill their host

temperate phages

usually dormant, can switch to lytic phase when time is right

lysogeny - integrase action

• only early genes transcribed

• phage lambda injects its DNA into host cell in linear form

• DNA re-circularises

• DNA integrates into host chromosome to create prophage - combined chromosome of bacterial and phage DNA

• this requires integrase enzyme

• circular chromosome of bacteriophage and bacterial chromosome have attachment-site sequences - matching recognition sites

• integrase binds to attachment-site sequences and aligns them to catalyse double-stranded break and rejoining to produce heteroduplex joints (around 7 nucleotides long) - bacteriophage DNA integrated into bacterial chromosome

• integrase dissociates after heteroduplex joints formed

lysogeny

• default pathway

• only early genes transcribed

• phage lambda has all essential genes arranged into 2 operons - left and right

• genes encoding N and cro protein transcribed - immediate early transcripts

• N anti-terminator protein - allows transcription to proceed beyond terminators, on leftward operon int, xis, cIII transcribed, on rightward operon, cII, O, P, Q, S, R transcribed

• cI gene between left and right promoters encodes lambda repressor - cII induces promoter protein (P_RE) for cI so that lambda produced, cIII stabilises cII so that it is more effective

• cII normally attacked and broken down by HflB proteases in bacteria so production inhibited - cIII prevents this

• lambda repressor inhibits expression of left and right operons so that they are not transcribed - no lytic pathway

• P_RM promoter (induced by lambda repressor) ensures continual cI transcription to ensure lambda repressor is continually produced

• if lambda levels are too high, the lambda repressor shuts down to save energy

lytic cycle

• both early and late genes transcribed

• phage lambda has all essential genes arranged into 2 operons - left and right

• genes encoding N and cro protein transcribed - immediate early transcripts

• N anti-terminator protein - allows transcription to proceed beyond terminators, on leftward operon int, xis, cIII transcribed, on rightward operon, cII, O, P, Q, S, R transcribed

• cro repressor - interferes with lambda binding

• whether lytic cycle occurs is a battle between lambda repressor and cro

• what lamba repressor does:

has highest affinity for O_R1 - preferential region for lambda to bind, overlaps with rightward promoter so once lambda has bound this represses lytic cycle genes

has second highest affinity for O_R2 - lambda binds to activate promoter for repressor maintenance - P_RM to ensure continual cI expression

only binds to O_R3 when lambda levels very high - this inhibits expression by locking action of RNA polymerase since it overlaps with the lambda repressor

• what cro does:

has highest affinity for O_R3 - prevents synthesis of lamba repressor

binds last to O_R1 - blocks its own promoter but by time cro levels are that high the lytic cycle is already complete

cII

• key in whether lysogeny or lytic pathway takes place

• can activate P_RE - lambda repressor produced so lysogeny takes place

• can prevent late gene anti-terminator Q from forming - Q unlocks expression of S and R so lytic pathway takes place

• activates pI - special promoter for integrase

what determines whether lysogeny/lytic cycle is favourable

• how many phage there are per bacterial cell

• how much food does the host have

• how healthy are bacterial hosts

• will not divide if don’t have sufficient food/are unhealthy so less bacterium produced for phage to infect - better to stay dormant, don’t want phages to outnumber bacterial cells

how bacteriophages correctly schedule transcription of genes to facilitate transitions through stages

• early genes expressed and produce protein

• this protein rises to sufficient levels so that it can act as a regulator for late genes - switches on expression of late genes

breaking lysogeny - induction of the prophage

• lambda repressor needs to be removed for process to be reversed - RecA has protease activity which cleaves lambda repressor into 2 halves - early gene expression in both directions

• RecA produced when there is DNA damage to host - activity activated by ssDNA

• RecA also cleaves lexA - lexA switches off repair genes needed in extreme circumstances

• integrase and exisase remove the prophage - excise phage DNA and recircularise it so that it re-enters lytic pathway

• right operon has structural genes which encode replication enzymes, coat protein synthesis, endolysin production