BC21C: Molecular Biology 1 Dr Sherline Brown

Bacteriophage Learning Objectives

To explain gene organization and expression in bacteriophage To describe the role of CI and Cro in transcription regulation To explain the factors determining lytic growth and lysogenic growth To describe anti-termination and retroregulation

Bacteriophage Course Outline

Morphology of bacteriophage Gene organization in bacteriophage Gene expression in bacteriophage Bacteriophage gene function The immnunity region Control of transcription by CI and Cro Factors determining lysogenic and lytic growth Lysogenic growth of bacteriophage Lytic growth of bacteriophage Antitermination and Retroregulation

Biology & Genetics of Bacteriophage

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

Recombination, gene regulation, protein folding

Bacteriophage Gene organization

Bacteriophage Gene organization

Lamda genome divided into functional units Morphogenesis Recombination Regulation Replication Lysis

Bacteriophage Gene organization

Bacteriophage Gene organization

Head & Tail
Genes code for structural protein of bacteriophage capsid Codes for terminase enzymes required to process rolling circle multimers into unit genome-length pieces during packaging

Bacteriophage Gene organization

Recombination
Code for Int and Xis genes which are required for integration of the bacteriophage into the bacterial host chromosome Codes for excision of bacteriophage from the bacterial host chromosome during induction Codes for other genes as well

Bacteriophage Gene organization

Regulation
Includes the immunity region as well as genes that are responsible for the genetic switch The Q antiterminator protein as well as the anti-Q RNA and PR`

Bacteriophage Gene organization

Bacteriophage Gene organization

Replication
This region includes 2 replication protein genes O and P and the origin of replication

Bacteriophage Gene organization

Lysis
There are 4 genes in the lysis region

Bacteriophage Gene organization

Some gene products and their function

Bacteriophage Gene Expression

Bacteriophage Gene Expression

Bacteriophage gene expression can be classified into 3 phases: Very early expression Early expression Late lytic or lysogenic expression

Bacteriophage Gene Expression

Bacteriophage Gene Expression

PL expresses the antiterminator protein N PR expresses the antirepressor protein cro PR` pauses after a short distance and no protein is expressed

Bacteriophage Gene Expression

Early expression depends on the action of the N protein The N anti-terminator causes expression from PR and PL to continue past transcription terminators

Bacteriophage Gene Expression

CII & CIII favour lysogenic growth O & P are required for replication Q favours the lytic growth

Bacteriophage Gene Expression

Only genes expressed are the lysis genes and the genes coding for head and tail proteins The anti-terminator protein Q is required for expression of these genes

Bacteriophage Gene Expression

At the same time, cro will Prevent any further gene expression from PR or PL

Bacteriophage Gene Expression

The only genes expressed are int and cI Once the bacteriophage has integrated into the bacterial host chromosome, cI is the only gene that will continue to be expressed

Bacteriophage The Immunity Region

Bacteriophage The Immunity Region

The immunity region of lambda contains: Three Promoters: Two Operators: Two Genes : PL, PRM, and PR` OL and OR c1 and cro

OR3

OR OR2

OR1

OR3 PRM

OR2

OR1 PR

Bacteriophage The Immunity Region

The immunity region of lambda contains: Three Promoters: PL, PRM, and PR` Two Operators: OL and OR Two Genes : c1 and cro

Bacteriophage The Immunity Region

The Operators
The operator is a specifc region of the DNA at the initial end of a gene where the repressor protein binds and blocks mRNA synthesis OL and OR consists of three operator sites to which either repressor cI or cro can bind
OL1 OR1 OL2 OL3 OR2 OR3

Bacteriophage The Immunity Region

The Operators
Each operator site is 17 bp long and has a center of dyad symmetry Each operator half site binds with one monomer of repressor or cro
OL1 OR1 OL2 OL3 OR2 OR3

Bacteriophage The Immunity Region

The Operators Repressors binds to each of the operator sites with different binding affinities OL1 > OL2 ~ OL3 OR1 > OR2 ~ OR3

Bacteriophage The Immunity Region

The Operators

CRO binds to each of the operator sites with different binding affinities OL3 > OL2 ~ OL1 OR3 > OR2 ~ OR1

Bacteriophage The Immunity Region

The operator-sites are not identical in sequence The OR3 operator-site is the only one with a T:A base pair at position 3 in both halves of the operator-sites. This is the site to which cro binds with the highest affinity
OL1 OR1

OL2 OL3 OR2

OR3

Bacteriophage The Immunity Region

The Promoters
A promoter region is the site on the DNA where RNA ploymerase binds and begins transcription

OL contains a single promoter PL

Bacteriophage The Immunity Region

The Promoters
PL directs transcription in a leftward direction through the N gene and eventually through the recombination region

PL is a strong promoter

Bacteriophage The Immunity Region

The Promoters OR contains two promoters PR and PRM PR directs transcription in a rightward direction through cro and eventually through the replication region PR is a strong promoter

Bacteriophage The Immunity Region

The Promoters PRM directs transcription in a leftward direction through cI PRM is a weak promoter

Bacteriophage The Immunity Region

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 The Immunity Region

Repressors
The repressor protein is a regulatory protein that binds to specific sites on DNA and blocks transcription, it is involved in negative control

Bacteriophage The Immunity Region

Repressors
Small polypeptide 27 kD N-terminal domain is the operator binding site N- terminal has a helix-turnhelix (HTH) motif C-terminal domain responsible for dimerization

Bacteriophage The Immunity Region

Repressors 2 domains joined by a connector Each of the operator sites in OR and OL binds a repressor dimer Each individual N terminal region contacts a half site

Bacteriophage The Immunity Region

Repressors The repressor is a H-T-H helix binding protein The 2 alpha helices of the HTH motif in adjacent repressor monomers contact each half of an operator site

Bacteriophage The Immunity Region

Repressors The amino acid sequence of the recognition helix makes contact with particular bases in the operator sequence that it recognises

Bacteriophage The Immunity Region

Repressors Cleavage of the domain can occur with the use of papain or the recA protein. This results in induction

Bacteriophage The Immunity Region

Repressors
Repressor binding to one operator increases the affinity for binding of a repressor dimer to the adjacent operator The affinity is 10 X greater for OL1 and OR1 than for the other operators so they bind first

Bacteriophage The Immunity Region Repressors

Co-orperativity allows the repressor to bind the OR1 and OR2 sites at a lower concentration

Bacteriophage The Immunity Region

Repressors
Coorperative binding increase the effective affinity of the repressor for the operator at physiological concentration Allows a lower concentration of repressor to achieve occupancy of the operator Why does this have serious consequences?

Bacteriophage The Immunity Region

Repressors
Repressor binding shows coorperativity OR1 ~ OR2 > OR3

Bacteriophage The Immunity Region

The Cro Protein Very small protein 66 amino acids Consists of a single domain which contains a helix-turnhelix DNA binding motif

Bacteriophage The Immunity Region

The Cro Protein Order of binding affinity is different OR3> OR2 ~ OR1

Bacteriophage The Immunity Region

The Cro Protein
Binding of the Cro protein to the operator site is similar to that of the repressor except Cro does not bind cooperatively Cro does not have amino terminal arms Cro forms a specific bond with T:A base pair at position 3 of the operator half site. This site is not recognized by lambda repressor

Bacteriophage The Immunity Region

Lambda repressor acts as a transcription activator whereas cro is unable to do so

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

CII is transcribed by PR and CIII is transcribed by PL CII binds upstream of PRE and stimulate the transcription of CI from PRE (CII can be described as?) Only when sufficient repressor has been made by PRE that it can bind to OR1/OR2 and direct its own synthesis

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

CII also activates PaQ Transcription from PaQ makes a short selfterminating transcript that function in antisense control of Q expression Transcription from PRE and PaQ serves to counteract the effects of cro and Q which promote lytic growth

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

Regulation of repressor synthesis in the lysogenic state

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

Role of cro protein in induction

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

Transcription continues beyond the sib site

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 Transcriptional Antitermination In

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)

Late stage (regulates synthesis of late genes including head and

tail proteins)

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