Viruses and phages

Several types of independent genetic units in bacteria

integrated

Plasmid vs. episome = lytic vs. lysogenic phage

an autonomous units as extrachromosomal genomes, self-replicating circular molecules

in a stable and characteristic number of copies

Plasmid:

free form

free form

integrated

integrated

die for transfer

conjugation

lysis

Two pathways of a phage: lytic and lysogeny

lysis

Prophage: latent form; as part of bacterial genome

Integration (repressor on an operator)gene expressionIn a cascade

immunity1. Excision2. Susceptible host3. Conditions of infection

infection

relief the repression

Conditions that favor lysogeny include: • Poor nutritional state of the host population (bacteria) • High multiplicity of infection (MOI). High ratio of infecting phage to host bacteria.

Lytic cycle of a phage

Goal: replicate a large number of progenyMethod: hijack host Tx/Tl machineries/apparatusResult: phage mRNAs are preferentially transcribedProcedure: 1. Infection

2. early event 3. late event

4. lysis

Early infection

Late infection

Make a populatio

n of

phage genome

Assembly

A cascade regulation in lytic phase: accompanied with the similar organization of the genetic map

small number of regulatory switches with cluster organization in a sequential expression order to Maximize economy

Gen

e ex

pre

ssio

n c

asca

de

in a

po

siti

ve

co

ntr

ol

man

ner

positive

(delayed early)

Immediate early

Begin at the phage genome starts to replicate

(i.e. assembly proteins)

Each set of genes are necessary for the expression of next genes

Cascade:

See next

Two types of regulatory event in lytic cascade (switch from early stage to next stage related to gene expression)

by new σ factor is made or RNA polymerase

by making antitermination factor (using the same promoter)

1. 2.

Previous gene expression continues at next stage

T7 phage

Phage genomes show functional clustering

Immediateearly

early late

T4 phage

Phage genomes show functional clustering

(165kb)

Numbered: essential genesThree-letter: non-essential genes (selective advantage)

Early: host RNA polymeraseMiddle: host RNA polymerase+MotA/AsiA (as activators to compensate for the deficiency of middle gene promoter at -30)

Late: host RNA polymerase + new synthetic sigma factor/modifier from middle phase

(initiation control)

Lambda (λ) phage: two lifestyles(anti-termination control

; positive control)

pN as an anti-terminator

pQ as an anti-terminator

host RNA polymerase

(Repressor control;Negative control)

Morphology 1. Double-stranded DNA genome, adopts both a linear and a circular form. 2. The protein coat, which protects the genome, is composed of a head and a tail. 15 different proteins, all encoded by the viral genome, form the protein coat.

How does anti-termination work in λ phage?

Gene clustering by functionalityImmediate early gene, N, encodes for

an anti-terminator

pN, an anti-terminator, allows the transcript to continue into the delayed early phase

(immediate early phase)

anti-termination(binds to nut site)

3 promoters

How does anti-termination work in λ phage? (delayed early phase)

λDNA join to form a circle after infection

PR

PR’

PL

+Q

-pQ: the transcript will stop at tR3 and is 194 bases long, known as 6S RNA

Anti-termination by pQ

Lysogeny: maintained by cI repressor

By denying RNA polymerase access to these promoters, a repressor protein (encoded by cI) prevents the phage genome from entering the lytic cycle.

cI repressor: 1. maintains the lysogenic state 2. provides immunity

an inefficient promoter for cI (lacking ribosome binding site). RM= repressor maintenancePRM

cI repressor

An autogenous circuit of cI repressorRepressor binding to the operators simultaneously blocks entry to the lytic cycle and promotes its own synthesis

Block lytic cycle Maintain lysogenic cycle

PRM

X X

repressorrepressor

*activate

To ensure the maintenance of lysogenic phase and immunity by cI autogenous circuit

2° infected phage only can enter lysogenic phase but not lytic phase

λvir:mutated OR or OL prevents repressor binding

cI expression is sensitive to its own existenceAbsence of cI repressor will stop the autogenous circuit of its expression

Immunity region

Mechanism

at aa. 111th/113th

UV

Structure of cI repressorDimeric structure of the repressor is crucial in maintaining lysogeny

Inducer binding siteChange conformation

~ ~

dimer simultaneously binds to DNA withhigh affinity

17bp palindromic sequence

Interaction of cI repressor and operator

Major groove34A

Allow binding to a successive major grooves of DNA

Contact with DNA bases

Cross over DNA

(protrusion from helix1)

contact with another face of DNA

Not contacted bases may be twisted to allow optimal contact between operator and repressore.g. phase 434 repressor contact 5 outmost of the half-site (G-C rich)The 3 inner bases (A-T rich) can easier result inwidening angle of two half-sites

DNA binding specificityAAs of helix make directly contact with bases of the operator

Chimera approach to prove the specificity of binding (protein-DNA).

Recognition helix:

Helix 3 forms several H-bonds with DNA basescontribute to binding specificity

Binding helix:

Helix2 forms H-bonds with phosphate backboneNot for specificity Also the ionic bonds contribute for interactions

Hydrophobic

H-bonds H-bonds

Hydrophobic

specificity

Keep relationship between helicesAffinity

K

contact with G in major groove and phosphate backboneMutation makes repressor affinity less than ~1000X

Cooperative DNA binding of repressor dimer

3 repressor-binding sites/operatorNo identical sequenceSeparated by 3-7 bp and AT-rich

Where to start binding O1 has strongest affinityO1 increases the affinity of O2

Usually O3 is not filled with repressor (no enough conc.)

Increases the effective affinity of repressor for the operator at

physiological conc. (or at lower conc)

OL1 and OR1 lie overlapping with RNA polymerase binding sites of PL and PR, respectively.Occupancy of OL1-OL2 and OR1-OR2 physically blocks access of RNA polymerase to the corresponding promoters.

Repressor interacts with RNA polymerase: an autogenous regulation of cI repressor

O2

turn/loopbetween helix 2 and 3

containing acidic patch to interact w/ basic region of RNA polymerase via electrostatic interaction

Stabilize the RNA polymerase binding to PRM promoter site to facilitate cI transcription(transition from closed complex to open complex)

O1

PRM

Protein-protein interaction can release energy that is used to help to initiate transcription

O3

establishmentstabilization-ve feedback

Both O1 and O2 mutations lead to virulence

How is the synthesis of repressor established in the first place?

cI: establishment and maintenance cII: maintenancecIII: maintenance positive regulators for initiation step of cI expression

(repression)pN

(+) via PRE promoter

(cIII protects cII degraded by HflA protease)

PRE promoter needs cII protein to facilitate RNA polymerase initiating transcription

cI

initiation

anti-cro

Inhibits translation of cro mRNA

cl protein expression

RNA transcribed to cI

Three related species of lysogenic bacteriophages have been studied, lambda, 434, and P22. A relatively small region of the phage genome contains all the genetic components of the on-off switch. In each of the three species of phages this region comprises two structural genes coding for the two regulator protein, cro and repressor, that operate the switch and the operator region (OR) on which they act. The operator region (OR) contains three protein binding sites – OR1, OR2 and OR3.

www.bioinfomaster.ulb.ac.be

The two genes are transcribed in opposite directions from their two promoters, which occupy opposite ends of the operator region. When RNA polymerase is bound to the right-hand promoter, cro is switched on, along with the early lytic genes that lie to the right of cro, and lysis results. When the polymerase is bound to the left-hand promoter, repressor is switched on, and cro and the lytic genes are repressed, and the cell survives as a lysogenic strain.

www.bioinfomaster.ulb.ac.be

The structures of their dimers are also known.

Lambda cro molecule Lambda repressor

www.bioinfomaster.ulb.ac.be

Lambda repressor/operator complex

Cro-DNA interactions

www.bioinfomaster.ulb.ac.be

Lysogenic phase. Repressor (red) and RNA polymerase (yellow) bound to the switch region in a lysogenic strain of E. coli. The repressor binds to OR1 and OR2, thereby turning off synthesis of cro. The repressor also works as an activator for its own synthesis by facilitating RNA-polymerase binding to the repressor promoter through its binding to OR2

Lytic phase. Synthesis of the cro protein turns off synthesis of the repressor, since cro binds to OR3 and blocks RNA-polymerase bindding to the repressor promoter. Transcription of the phage genes to the right can now occur

www.bioinfomaster.ulb.ac.be

Why does RNA polymerase need cII protein to take place transcription?

PRE promoter has a lack of -35 consensus seq.

-45 -25 -12 +13

cII regulator binds to a region extending from -25 to -45Only when it is added, RNA polymerase can binds to PRE promoter

Cis-acting mutation affects the establishment of lysogenySimilar phenotype as cII or cIII mutant

(trans-acting mutants)

Lysogenic phase of λ phage

All immediate early and delayed early genes are

turn off

Integration into genome

The role of cII in lysogenic phase(establishment)

Acts at PRE to transcribe cIActs at PI to transcribe int (for integration)Acts at Panti-Q to transcribe antisense RNA of Q mRNA, ensures degradation of Q

The role of cI in lysogenic phase(maintenance)

Autogenous circus to turn off all genesvia PRM and OR ， OL

Block PR and PL gene expression

The entering lytic cycle:N: anti-termination; cro: repressor of lysogeny

Acts at OR3 to prevent RNA poly binding to PRM,

blocking maintenance of cI repressor. Acts at OR/OL to prevent RNA poly from expressingimmediate early genes, blocking repressor establishment.

The role of cro repressor: dimer/9kD each

Mechanism is similar to cI repressor

A tug of war between cI and croThe occupancy of repressor (cI) and cro at operators

cII is the judge

Role of cII

Mechanism of Lysogen Induction by UV Light UV irradiation damages DNA and thus activates E. coli’s DNA repair systems. One of the DNA repair proteins, RecA, normally functions to repair double-stranded DNA breaks. However, RecA also acts as a coprotease to facilitate the cleavage of CI (lambda repressor). CI itself has latent autoproteolytic activity, but requires the RecA coprotease to stimulate this activity. Lysogenic induction would proceed as follows:

• UV light damages DNA and activates RecA • RecA binds to CI • The RecA-CI complex cleaves CI (autoproteolysis) • In the absence of CI, transcription from the right and left promoters (PR and PL) resumes. • CRO is produced, which inhibits the synthesis of CI and stimulates expression of lytic genes. • Phage particles are formed and the cell is lysed.

The x-ray structures of DNA-binding domain of the lambda cro and repressor are known.

The N-terminal domain of lambda repressor, which binds DNA, contains 92 amino acid residues folded into fives helices. Two of these, 2 (blue) and 3 (red) form a helix-turn-helix motif with a very similar structure to that of lambda cro. The complete repressor monomer contains in addition a larger C-terminal domain.

www.bioinfomaster.ulb.ac.be

In the case of identical domains, the DNA recognition site comprises two half sites that are either direct or inverse (palindromic) repeats of each other.The base pair separation between the two half sites is specific to each protein; it depends upon the linker region between the motifs when these belong to the same sequence, and on the protein-protein dimerization interface otherwise.

www.bioinfomaster.ulb.ac.be

Oct1 pou domainPDBcode: 1octR = 3.0 Å R factor = 0.237

Association of two different DNA-binding motifs (here, homodomain and classic HTH type in oct1 pou protein). The target site encompasses 2 major grooves and one minor.

www.bioinfomaster.ulb.ac.be

Fos (mauve) – Jun (red) –Nfat (blue)PDBcode: 1a02R = 2.7 Å R factor = 0.246

Association between two different DNA-binding domain, one leucine zipper and a more complicated one. The target site becomes 3 major grooves and one minor.

www.bioinfomaster.ulb.ac.be