Bacteriophage lambda PDF

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Lecturer: Dr. Cain
On the 7th October 2019....

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BIOC0005 – Molecular Biology Bacteriophage Lambda (Dr. Cain) 7/10/19

BACTERIOPHAGE History -One of the first model organisms used because very accessible genome (small) -Understandable – fully sequenced (promoter and operator sites) -Accessible in the 50s bc many different mutations were identified -First organism to have end of chromosomes understood -1st organism to have genome completely sequenced (1982) -Important in study of transcriptional termination Bacteriophages -Virus that infects bacterial cells -Simple entities, nucleic acid wrapped up in protein -Minimal functions:  Protect nucleic acid (wrapped up in protein coat)  Delivery of nucleic acid inside host cell  Convert infected cell into phage-producing factory  Release of phage into environment (for next infection stage) -Limited strategies, bc small -> no physical space for DNA to replicate or develop other complex processes CAREFUL = phage is pl and sg, when talking about phages = different spp Phage classification On how they look:  Icosahedral head attached to a tail (with fibres, uses to adhere to cell structure), tail is optional  Filamentous phage, long DNA wrapped up in protein. Less limited, to make DNA longer makes more protein to wrap around it (protection) On type nucleic acid: - DNA or RNA - Double stranded or single stranded - Circular or linear - Etc Phage life cycles: lytic growth 1st: Phage particle makes contact with cell, normally tail fibres make contact cell -> bind receptor in cell surface (not made by bacteria for bacteriophage, the phage takes advantage of it)

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2nd: DNA/RNA inside host cell, depending on str of phage on how it’s done (eg, injection through tail). Though that with filamentous phages, the nucleic acids (brief period) become susceptible to nucleases outside cell -> uncoat to shed its coat protein -> taken up by cell (similar to transformation)

3rd: Early stage of development, host enzymes to read DNA to make proteins and replicate DNA. Many phage particles don’t contain any phage proteins, so need to use host enzymes to produce phage proteins. Cascade, some proteins needed in later phase are produced to trigger final phase (development of phage genome)

4th: Late development, genomes, heads and tails are made. DNA packaged into heads and tails attached.

5th: Lytic event, cell is broken to release phage progeny. Some filamentous phages don’t break the cell open, instead they extrude themselves through cell membrane (keep host cell intact)

Phage life cycles: lysogenic growth -Adhesion phase -Nucleic acid injected into host cell -Phage DNA is incorporated into bacterial genome -> stable prophage -> lysogen (=bacterial cell), every time it replicates, phage DNA gets replicated too -Once phage DNA is incorporated into bacterial genome, bacteria are protected and immunised from further infection (big advantage) -Induction event = phage DNA is released from bacterial chromosome and back into lytic phase if conditions change (good conditions = lytic cycle; unfavourable conditions = lysogenic cycle) -Slower mechanism of reproduction, but advantages [see later]

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Basic terminology -Virulent: ALWAYS undergo lytic cycle -Temperate: choose between 2 cycle (majority) -Lysogen, bacterial cell with phage DNA integrated in its chromosome -Prophage, incorporated bit of DNA in lysogen (possibility to form phage again in future) -Immunity, characteristic that lysogen has that stops it from being infected by further bacteria once it has prophage integrated -Induction event, what happens to remove prophage from bacterial chromosome to send it down a lytic pathway after a period of being in lysogenic state

Lysis or lysogeny -Depends on conditions of host bacterial cell -Lysogeny favoured by: -> Low levels of nutrients (not happily growing) -> High MOI (Multiplicity Of Infection = hosts ratio of bacterial cells to phage particles) = more phage than bacterial. If phage infects bacterial colony growing happily, it’ll infect rest of them quickly (lytic cycle) and run out of hosts -> If infects bacteria about to go dormant, it would get lost bc phage needs bacteria to be active, so the phage integrates into genome (“hiding”)

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Bacteria can go through period of dormancy, where shut themselves down, stop protein synthesis, form coat around themselves to stop them from dehydrating, until conditions are improved.

Identifying lysis/lysogeny in LAB -> grow complete lawn of bacteria on agar plate (not look for colonies!!) -> introduce phage -> every time it infects = plaque: -Clear = virulent phage = every time it infects bacteria, it kills them (clear spot) -Turbid = start of lytic route (kill bacteria) -> at some point, one of those bacterial cells goes down lysogenic pathway -> lysogens start growing again (overgrow area where bacteria died) => less bacterial

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BACTERIOPHAGE LAMBDA -Icosahedral head with tail attached that contact E. coli cells -Linear ds DNA (48,514 bp) -12 bp cohesive ends (at either end of linear strand of DNA) that stick out of the ds molecule [restriction enzymes with overlapping restriction sites -> the single stranded bits of DNA Hbond together to reform molecules] -> when it’s injected in bacterial host cell DNA goes from linear to circular (bc 12bp overhangs form H-bonds together forming a circular molecule. Bacterial DNA ligase ligates phosphodiester backbone]. -Linear in phage particle, circular in host cell (to be able to replicate) -46 genes identified (know what they do, know promoters, terminators and operator sites are) Lambda genetic map

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Genes clustered in regions that relate to function (all expressed at same time): -1 long transcript for head and tail genes -Integration and excision = integrated in lysogeny -Recombination genes -Control region = where decision is made to whether go down lytic or lysogenic pathway, genes expressed important for the switch -2 genes for DNA replication -Gene for late control -Lysis of cell once phage particles have been made -cos site (cohesive end site) = where linear genome is circularised -> joint happens between S and Head genes. If DNA doesn’t get circularised = Pr transcript doesn’t happen (important for lytic cycle)

LYTIC CASCADE Lytic development controlled by a cascade -Cascade of gene expression -When first injected into host cell, normally no phage proteins -> relying on host proteins -Host RNA pol is required for expression for initial phage genes -> recognises promoter (similar to bacterial promoter) -> few genes produced = one of them is a positive transcriptional regulator for next set of expression -Positive tr regulator could express: -> Own RNA pol that recognises very different promoter -> Sigma factor = binds to host RNA promoter that causes recognise a different type of phage specific promoter 5

-> Antitermination factor = causes host RNA pol to ignore terminator, so initially produces small transcript and instead of stopping, carries onto next phage expression -One of the above will turn on the next phase of phage expression = immediate late phase = another regulator is produced (turns on late phase) -Late phase of expression = another regulator that changes how genes are expressed -> transcription of late genes

Lytic cascade – key points -Early genes are ALWAYS transcribed by host RNA pol (inc regulators for delayed early expression) -Delayed early expression inc regulators for transcription of late genes -Expression of groups of genes occurs in an ORDERED matter Lytic cycle: EARLY TRANSCRIPTION -2 promoters in control region of lambda genome => left promoter ( PL) and right promoter (PR). Host RNA pol recognises both promoters: -PR transcribes rightwards and makes cro protein -> tR1 terminator (transcript stops) -PL transcribes leftwards and makes N protein (= regulator, anti-terminator) -> binds to a nut site (N-utilisation site) that lies between the promoter and the terminator -> when host RNA pol transcribes nut site, picks up N protein -> changes -> ignores terminator -> doesn’t terminate = longer transcripts

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Lytic cycle: GENE EXPRESSION -2 promoters just before N and cro proteins -Immediate early phase = get small transcripts to make proteins N and cro (black lines) -Delayed early phase = N allows host RNA pol to ignore first terminator, same transcript continues around genome (left) until it reaches different terminator -> expression of other genes => some genes required for lysis, others for lysogeny. We need expression of early genes + delayed early genes regardless of which pathway we are following. Once all genes expressed, cell can determine which pathway is followed. -Q (delayed early protein) = next regulator protein, turns on expression of late genes (controlled by antitermination transcript made from PR) -Late phase = host RNA pol binds to PR’ -> makes 194 bp transcript (no functional genes). Expression of Q leads to a second antitermination event -> binds to Q-utilisation site (qut), causing host RNA pol to keep transcription going on => HEAD AND TAIL PROTEINS = committed to lytic pathway

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DNA replication -Once phage is inside host, turns on its genes -> if lytic cycle -> lots of copies of genome for packaging into new particles. -2 different phases: 1st: classic replication of circular molecule ( theta replication): makes more molecules that can be expressed inside the bacterial cell: -melting of ds molecule at ORI -> formation of primers, extended at opposite directions. 2 daughter molecules formed (each of the daughter contains original and newly synthesised daughter strand) -Requires 2 of the phage proteins (O and P) and host RNA pol III. -O pr binds to origin -P pr goes out into the cytoplasm, recruits bacterial helicase, which binds to O pr (at origin) -Initiates replication process -> unwinding of DNA + recruiting of all bacterial replication enzymes to synthesise new strand. nd 2 : rolling circle replication = in order to package DNA into phage heads, has to be linearised. -NO melting of two strands, nick at origin = one of the strands if broken -Produces 3’ OH end = anchor point, what we need on a primer 8

-Host DNA pol synthesises new strand starting from the nick -> reads round and round to make long molecule that contains multiple linear copies of phage genome = concatemers of phage genome (long continuous DNA molecule with multiple copies of same DNA seq linked in series) -Double stranded DNA is needed for the phage!!!

-Lots of head and tail proteins produce that form actual phage particle, but only 4 head proteins coming together to form initial head sphere -Terminase complex = 2 proteins ( NU1 and A), which are involved in cutting DNA in cos sites to create single 48k bp linear molecule + act as DNA translocase (push DNA into head particle). Associate with neck region of the head that’s forming and recognise a cos site in linear molecule -> cleave it -> hold onto it -> push DNA into head (conformational changes that go from rough spherical head to icosahedral shape) until they get to second cos site -> cleave that second one (and on and on) -> single stranded DNA ends stick out from phage head = associated with binding of tail to head structure -Cell lysed -> released out into the environment -> infect other hosts

LYSOGENY -Delayed early genes -> cII and cIII proteins = turn on lysogeny, activating cI gene (lambda repressor), if it’s made in sufficient quantities = cI repressor binds and turns off lytic cascade -cro = repressor, represses lysogeny (negative regulator)

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-Lytic cascade always starts but depending on concentration on different proteins (cro vs cI) as to whether the lytic cascade can go as further as the head and tail proteins, or repressor turns off lytic cascade

Lysogeny and the lambda repressor -3 promoters = PL, PR and PR’ -> associated with operators (OL and OR) [operator = seq of DNA that can bind a regulator, things binding to an operator can turn on-off a promoter = “switch”] -nut site = where N protein binds to the N-utilisation site (where is picked up by host RNA pol), causing terminators to be ignored -N and cro = immediate early genes -After first terminators, expression of cIII (leftwards) and cII (rightwards) = activators that turn on expression of cI (which codes for lambda repressor) -PRM and PRE are both promoters for the cI repressor -> PRE is turned on first (by cII and cIII, positevly regulate) -> produces transcript in left direction from promoter for repressor establishment -> makes transcript that encodes lambda repressor -> repressor binds to operators associated with PL and PR = repressing them. -Binding to OR activates PRM, which turns on promoter for repressor maintenance (only promoter that is active if cell goes into lysogenic state) -> cI auto-regulates its own expression with PRM -PRE only used initially in turning on expression of cI, but PRM is used longer term (if lysogeny is the outcome of infection)

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Repressor molecule (cI) provides immunity to lysogen -Host RNA pol expresses cI gene from PRM -cI acts as a dimer -> binds at operators (turning off PR and PL). In lysogen, loads of repressor molecules already. If another phage injects chromosome inside cell, immediately -> repressor dimer binds to operators of that incoming phage and turns off expression before it even starts -> IMMUNE OF FURTHER PHAGE INFECTION -Wouldn’t be immune to repression of completely unrelated species Lambda repressor -27kD protein -2 distinct domains: -> N-terminal domain binds to DNA -> C-terminal domain: interacts with another C-terminal domain forming dimer -Linker region – holds both domains together (=2 aa). Important! In induction event, C1 gets broken down via linker region -> decreased affinity for DNA significantly (at least 10 times) -> lambda DNA released from repressor

-DIMERISATION! = puts 2 N-terminals in such position (orientation) that have strong affinity to bind DNA at operator site!!!!

Entry into lysogeny -PRE = turned on in presence of cII (delayed early protein) -> required bc in normal circumstances, host RNA pol does NOT bind strongly to PRE (weak match) -> so cII binds in 11

region -25 to -40 in relation to transcription start site -> host RNA pol recognises weak -10 region of promoter and turns it on = POSITIVE TRANSCRIPTION REGULATOR -BUT, cII is frequently degraded by host proteases => cIII protects it by binding to it -When cell is growing, high concentration of proteases -> higher chances of cII being degraded = NO turning on expression of PRE -If cII survives -> turns on expression of PRE -> transcript produced = codes for cI (expressed for lambda repressor protein) (cII has mainly 4 functions -> promoting lysogeny) -Because the transcript you get is complementary to mRNA you can get ds mRNA (hybridisation) = do NOT see translation -> So, by turning on PRE, you stimulate the expression of lambda repressor from cI AND turn down expression of cro (bc the transcripts will get hybridised, so cannot be translated into cro protein) -Turns on another promoter (PI – turns on expression of integrase gene) -Integrase – needed for integration of DNA into bacterial chromosome -Turns on another promoter = P anti-Q => turns on expression of anti-sense transcript of Q -> will hybridise with Q transcript -> stops transcription of Q protein (which is required for lysis) = turns down expression of Q protein

Summary of lysogeny

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Lambda insertion -Site-specific recombination = lambda genome ALWAYS integrated at same point into bacterial chromosome = att sites (att = attachment) in cytoplasm: ->attP = site in phage ->attB = site in bacterial chromosome -Common seqs in each of these sites (called O) -> cross-over in O sequence -Flanking O seq there are 2 distinct seqs -> P and P’ (phage) and B and B’ (bacteria) -When integrated -> 2 new att sites formed (still containing O seq in middle) -attL and attR = attachment sites when the lambda chr is integrated in bacterial chromosome (we get excision from these, instead of attachment) -Always required -> IHF is host protein (always required) = Integration Host Factor + Int protein (integrase) -Excision requires an additional protein -> Xis = required in the case of an induction event (after lysogeny is broken) -In which direction the site-specific recombination goes in depends on seqs and proteins that are present

Site-specific recombination -In O regions (same in phage and bacteria) -Single stranded overhanging cuts in cos sites -> join one cut end from each = recombinant junctions -Happens in complex = intersome => integration host factor ( IHF) acts as scaffold when bound to DNA = DNA wraps around it and exposes the O seq to proteins that cut DNA [integrases that make cuts and re-joins DNA, coded by lambda int gene] -Similar mechanism between integrase and topoisomerase: - Topoisomerase = cuts DNA, unwinds DNA and re-joins cut in same place -Integrase = cuts DNA and re-joins into another molecule (coming from bacterial chr) -IHF + O seq + integrase -> recruits bacterial DNA, binds to complex, 2 cuts made, re-joining of 2 ends of molecule to form junction

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IN REVERSE: -Same, but Xis protein required to recognise the new sites (different flanking regions, different sequences) The operator region -Associated with promoters -Made up of 3 binding sites: each one made up from 2 half sites. Each one N-teriminal region of lambda repressor interacts with one half region of operator. -If sth is bound to first position of operator -> RNA pol cannot bind -> NO transcription -cro binds to same operators as lambda repressor, but it’s a much smaller protein. Also dimerises (not 2 domains). 2 proteins have similar helix-turn-helix binding domains (so they can bind same regions of DNA, but differences make them bind to different sites with different levels of affinity) -Lambda repressor has high affinity for OR1/OL1. Has cooperativity = if there is a dimer bound in O1 it drastically increases affinity of binding of second dimer to O2. NO cooperativity in O3 nor in cro protein! => as soon as lambda repressor binds to O1, it immediately binds to O2 = turns on promoter for repressor maintenance (PRM) -RNA pol binding site only overlaps O3 -If high expression of lambda repressor -> bind to OR3 -> turns off own promoter -> stop making protein (it’s its own promoter) -> concentration reduces -> let’s go of binding site -> expression starts again (releases promoter) -High expression of cro (=repressor for lysogeny) -> turns off promoter RM -> stops expression for lambda repressor (stops expression from PRM) -> Also binds to other operator binding sites, but at higher concentrations -> binds to O3s first and then 1 and 2 at higher concentrations (so turns off PR and PL too, but once cell is committed to lytic pathway = once there are many late delayed genes, lots of Q proteins)

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Summary of lytic cycle

Remember – lytic cycle initiation -When cro binds to OR3 it prevents RNA pol from binding to PRM and blocks the maintenance of lambda repressor protein -When cro binds to other operator sites it prevents RNA pol from binding to PL and PR (when cell is committed to lytic cycle) and turns off early gene expression

Lysis or lysogeny? -Comes down to whether what happens in operator!!!! -Lots of cro protein or lots of lambda repressor protein? -Deciding effect as to whther cro or lambda repressor dominates comes down to concentration of cII, bc required to turn on expression of cI gene (=encodes for lambda repressor) -If cII is degraded initially never see expression of PRE -> cro binding at operators -> depends on conditions cell 15

KEY POINTS TO REMEMBER -ALWAYS see expression of immediate early expression of N and cro -ALWAYS see delayed early expression of C2R and PQ etc -Delayed early stage when both cro and repressor are being expressed is common to both lysogeny and lytic cycle -It’s what happens after that that chang...