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Bacteriophage Lambda
Bacteriophage Lambda
Bacteriophage
What is a bacteriophage? A bacteriophage is a virus that infects prokaryotic cells. Affect enteric bacteria such as E. coli and Salmonella typhimurium
Bacteriophage
Why study bacteriophages ? Viruses represent the ultimate in parasites Unique system for studying regulation of gene expression as well as protein - protein interaction
Bacteriophage
Bacteriophages are diverse Can have dsRNA or ss RNA genome Can have ss DNA or dsDNA genome Best studied are those with dsDNA genome
Bacteriophage
A phage can either be virulent or temperate Virulent phages: lyse or kill their hosts after infection (lytic)
Bacteriophage
Tempererate phages: can achieve a state where their genome replicates along with the host genome without killing it (lysogenic)
Bacteriophage
Lytic growth: you have replication of phage DNA and synthesis of new coat proteins. They combine to form new page particles that are released by lysis of host cell
Bacteriophage
Lysogenic growth: Involves integration of phage DNA into the chromosome of the host cell where it replicates during cell division
Bacteriophage
Prophage: integrated phage Lysogen: bacterium harbouring the prophage Lysogenic bacteria has immunity against further infection from a similar bacteriophage
Bacteriophage
Induction: switch from lysogenic to lytic pathway Lysogeny is of ecological importance Best studied temperate phage is lambda Lambda has both lytic and lysogenic pathway
Bacteriophage Morphology
Bacteriophage Morphology
Lambda virion is similar to that of other tailed bacteriophages however no tail fibers are present Genome consists of a linear ds DNA molecule
Bacteriophage Morphology
Genome consists of a linear ds DNA molecule Size 48, 502 base pairs The 5` terminus of each strand has a single tail (12 nt long) These single stranded ends are complementary (ends are said to be cohesive)
Bacteriophage
When the two ends of the DNA are free in the host cell they associate (the cos site) DNA is ligated to form a double stranded circle
Bacteriophage
Best studied host- virus system Led to increase in knowledge about phage and their development Uncovered unknown information about host function
OR3
OR OR2
OR1
OR3 PRM
OR2
OR1 PR
CRO binds to each of the operator sites with different binding affinities OL3 > OL2 ~ OL1 OR3 > OR2 ~ OR1
OR3
PL is a strong promoter
PRM overlaps OR2 by 2 base pairs PR overlaps OR2 by 3 base pairs Difference allows repressor bound to OR2 to activate transcription from PRM but repress transcription from PR
How are these promoters controlled? This is where the repressor comes in
Bacteriophage
Control of transcription at OR by the repressor
OR contains 2 promoters: PR and PRM
RNA polymerase will readily bind to PR and initiate transcription in a rightward direction through Cro RNA polymerase may under the right conditions bind to PRM and initiate leftward transcription through the CI gene The ability of RNA polymerase to bind to PR and PRM and initiate transcription depends on the conc of Cro and CI
Bacteriophage
Repressor bound at OR2 blocks expression at PR but activates PRM. This is due to the fact that: PR overlaps OR2 by 3 bp while PRM overlaps OR2 by 3 bp Repressor can form contact with RNA polymerase that increases its binding affinity for a promoter
Acts as a transcription activator
Bacteriophage
Repressor bound at OR3 blocks expression at PRm.
Bacteriophage
Bacteriophage Lysogeny
Bacteriophage Lysogeny
Establishment of lysogeny
CI also activates PRM and directs more of its own synthesis CII also activates PI PI allows the transcription of the Int gene The Int enzyme integrates the lambda DNA into the bacterial DNA
Bacteriophage Lysogeny
Establishment of lysogeny
CIII inhibits cellular proteases that degrade CII If CIII is absent CII will be rapidly degraded and no lysogen will be formed
Bacteriophage Lysogeny
Establishment of lysogeny
The level of CII in the cell is dependent on two proteases HflA and HflB (FtsH) The level of the host proteases is dependent on the growth state of the bacteria Well fed E. coli is high in proteases Starving E. coli are low in proteases
Bacteriophage Lysogeny
Establishment of lysogeny
The level of CII in the cell depends on: The gene dosage of CII and CIII in the cell If lambda infects E.coli at a multiplicity of infection (moi) of 1 phage /cell, insufficient CII will be expressed to activate transcription and lysogenic growth If moi is 10 phage/cell the amount of CII and CIII will increase The extra CIII will protect CII
Bacteriophage Lysogeny
Establishment of lysogeny
The dependence of lysogeny on moi in the cells explains why bacteriophage lambda forms turbid plaques The dependence of lysogeny on moi also explains why induction is irreversible
Bacteriophage
Integration of
Bacteriophage lambda circularizes after infection Int promotes recombination between the attachment sequence (attP) on the phage protein and a site on the bacterial DNA (attB)
Bacteriophage
Integration of
The recombination promoted by Int is site specific This site specific recombination is nonhomologous, only a short region of homology is present (7 bp) In light of this Int is needed because it recognizes attP and attB
Bacteriophage
Maintenance of
After the lysogen has been formed the CI repressor is one of the few genes to be transcribed In the prophage state the CI gene is transcribed by PRM instead of PRE
Bacteriophage
Maintenance of
Regulation of repressor synthesis in the lysogenic state
CI repressor is functional only as a dimer At very low concentrations of CI polypeptides dimers cannot be formed At optimal concentration CI repressor binds to OR1 then OR2. If conc is too high it will bind to OR3 and regulate its own synthesis
Bacteriophage
Maintenance of
Immunity to superinfection The CI gene regulates the transcription of any other lambda phage infecting the lysogenic cell by binding to the operators of the phage Hence immunity to lambda super infection However similar phages with a different operator sequence can infect the lysogen
Bacteriophage
Induction
will remain a prophage until the cell is damaged The RecA when complexed with ssDNA can activate the protease activites of other proteins of the host. Brings about the autoclevage of LexA protein . repressor is similar to LexA. Proteolytic cleavage of the CI repressor also accurs
Bacteriophage
Induction
Dimerizationis lost Repressor drops off operators Transcription initiated from PL and PR Lytic cyle
Bacteriophage
Induction
Role of cro protein in induction
Cro gene product is one of the first proteins to be produced after induction Cro prevents the synthesis of more repressor Cro binds first to OR3 then OR2 preventing the repressor from binding to OR2 Cro binds to OL3 prevents repressor binding to OL PL is no longer repressed
Bacteriophage
Bacteriophage
Exision
Transcription from PL and PR can now take place PL transcribes Int and xis Exision requires both genes because the recombination occurs between different sequences from those used for integration Integration required combination between attB and attP
Bacteriophage
Exision
Exision requires recombination between the hybrid attP-attB sequences that exist at the junction between the prophage DNA and the chromosomal DNA This sequence is different from those at attP or attB Int alone cannot exise the DNA, it can only take place when xis is present The molecular basis for this is described as retroregulation O and P genes are transcribed by PR (what process is this required for)
Bacteriophage
Exision
Bacteriophage
Lytic growth
Very early expression
When bacteriophage has infected a cell and injected its DNA genome will be transcribed by host RNA polymerase from 3 strong promoters: PL, PR and PR`
The first genes to be expressed after bacteriophage lambda infection are N and cro
Bacteriophage
Lytic growth
Early Expression The early transcript of PR codes for
Cro CII O& P Q anti-repressor transcription activator required for lysogenic growth required for replication of the bacteriophage anti-terminator protein required for late gene expression
Bacteriophage
Lytic growth
Early Expression The early transcript of PL codes for CIII Xis Int required to protect CII required for exision of the prophage required for integration of the prophage
Bacteriophage
Lytic growth
Early expression
sib is an RNase II processing site Any transcript which passes through sib will form a hairpin structure that will be cleaved by RNase III The free 3` ends will be degraded by exonuclease. Int and Xis will be degraded before it can be translated This type of regulation is called retroregulation
Bacteriophage
Lytic growth
Late Expresssion
Late gene expression depends on the expression of Q and cro proteins Q allows PR` to transcribe the lysis genes (S, R, Rz) and the capsid proteins (Nu1, A, W etc) Cro binds to the 3 operator sites in OL and OR Cro represses all further transcription from PL and PR Prevents further expression of N, CII, CIII so lysogenic growth cannot occur
Bacteriophage
Steps leading to lytic and lysogenic development
Bacteriophage
Steps leading to lytic and lysogenic development
Bacteriophage Antitermination
The two antiterminators are N and Q uses antitermination regulation at two stages of its development Early Stage (regulates synthesis of recombination and replication
functions)
Bacteriophage
The N protein
When DNA infects a cell 2 rRNA are transcribed that synthesizes the N and Cro genes. Then it stops Initial transcription initiated at PR promoter terminates at TR` One of the sequences transcribed into RNA is nutR (N utilization rightward)
Bacteriophage
The N protein
N will bind to RNA polymerase after the nutR region has been transcribed. RNA polymerase with N added will transcribe past the TR` site Similar events occur on the left side
Bacteriophage
The N protein
The protein binds to the nut site sequence on the mRNA rather than on the DNA The nut sites consists of a sequence called BoxB that N binds to N changes its conformation after binding to BoxB In this state it is able to bind to RNA polymerase and prevent termination
Bacteriophage
The N protein
The host proteins collaborate with N in causing antitermination These are called nus protein Nus proteins are (nusA, nusB, nusE, nusG)
Bacteriophage
The Q protein
Q is a gene under the influence of the N anti-terminator Q allows transcription from the late promoter PR` to proceed through terminators into downstream genes Q loads on to RNA polymerase in response to a sequence (QBE or qut) located close the promoter
Bacteriophage
The Q protein
The qut sequence is not found totally in the mRNA, some of the required sequence is in the DNA (not transcribed) In the absence of Q, the polymerase binds to PR` and initiate transcription It pauses after 16-17 bases have been transcribed and terminates at TR`
Bacteriophage
The Q protein
In the presence of Q, Q binds to qut once the polymerase has left the promoter and transfers to the paused polymerase, allowing it to transcribe through TR`
Bacteriophage
Retroregulation
The int mRNA initiated at PL is degraded by cellular nucleases The int mRNA initiated at PI is stable and can be translated into integrase protein RNA initiated at PI stops at a terminator 300nt after the end of the int gene. It forms a stem loop with 6 uridine bases
Bacteriophage
Retroregulation
Bacteriophage
Retroregulation
When RNA synthesis initiated at PL, the RNA polymerase is modified by N and goes beyond the terminator at the end of int gene
This longer mRNA forms a stem that is the substrate for nucleases, hence it is degraded .
Bacteriophage
Retroregulation
Bacteriophage
Retroregulation
When the phage genome is integrated in to the host chromosome the site causing the degradation is removed from the end of the int gene Int mRNA can be made from PL