Viruses and phages

Several types of independent genetic units in bacteria lysis die for transfer free form integrated integrated free form integrated an autonomous units as extrachromosomal genomes, self-replicating circular molecules in a stable and characteristic number of copies conjugation Plasmid: Plasmid vs. episome = lytic vs. lysogenic phage

Two pathways of a phage: lytic and lysogeny infection gene expression In a cascade Integration (repressor on an operator) Prophage: latent form; as part of bacterial genome lysis Excision Susceptible host Conditions of infection immunity 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 Early infection Late infection Goal: replicate a large number of progeny Method: hijack host Tx/Tl machineries/apparatus Result: phage mRNAs are preferentially transcribed Procedure: 1. Infection 2. early event 3. late event 4. lysis Make a population of phage genome Early infection Late infection Assembly

A cascade regulation in lytic phase Immediate early A cascade regulation in lytic phase : accompanied with the similar organization of the genetic map Cascade: positive Each set of genes are necessary for the expression of next genes small number of regulatory switches with cluster organization in a sequential expression order to Maximize economy See next (delayed early) Gene expression cascade in a positive control manner Begin at the phage genome starts to replicate (i.e. assembly proteins)

Two types of regulatory event in lytic cascade (switch from early stage to next stage related to gene expression) 1. by new σ factor is made or RNA polymerase by making antitermination factor (using the same promoter) 2. Previous gene expression continues at next stage

Phage genomes show functional clustering T7 phage Immediate early early late

T4 phage Phage genomes show functional clustering (initiation control) (165kb) Numbered: essential genes Three-letter: non-essential genes (selective advantage) Early: host RNA polymerase Middle: 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

(anti-termination control Lambda (λ) phage: two lifestyles (anti-termination control ; positive control) (Repressor control; Negative control) host RNA polymerase 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. pN as an anti-terminator pQ as an anti-terminator

Immediate early gene, N, encodes for How does anti-termination work in λ phage? (immediate early phase) Immediate early gene, N, encodes for an anti-terminator Gene clustering by functionality 3 promoters anti-termination (binds to nut site) pN, an anti-terminator, allows the transcript to continue into the delayed early phase

How does anti-termination work in λ phage? (delayed early phase) λDNA join to form a circle after infection Anti-termination by pQ PL PR -pQ: the transcript will stop at tR3 and is 194 bases long, known as 6S RNA PR’ +Q

prevents the phage genome from entering the lytic cycle. 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 cI repressor PRM an inefficient promoter for cI (lacking ribosome binding site). RM= repressor maintenance

An autogenous circuit of cI repressor Repressor binding to the operators simultaneously blocks entry to the lytic cycle and promotes its own synthesis 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 Maintain lysogenic cycle Block lytic cycle λvir: mutated OR or OL prevents repressor binding *activate repressor repressor PRM Immunity region X X cI expression is sensitive to its own existence Absence of cI repressor will stop the autogenous circuit of its expression

Mechanism Structure of cI repressor ~ UV Dimeric structure of the repressor is crucial in maintaining lysogeny Structure of cI repressor at aa. 111th/113th ~ Inducer binding site Change conformation UV dimer simultaneously binds to DNA with high affinity

contact with another face of DNA Interaction of cI repressor and operator Not contacted bases may be twisted to allow optimal contact between operator and repressor e.g. phase 434 repressor contact 5 outmost of the half-site (G-C rich) The 3 inner bases (A-T rich) can easier result in widening angle of two half-sites Allow binding to a successive major grooves of DNA Major groove Cross over DNA 34A contact with another face of DNA (protrusion from helix1) Contact with DNA bases 17bp palindromic sequence

DNA binding specificity AAs 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 bases contribute to binding specificity Binding helix: Helix2 forms H-bonds with phosphate backbone Not for specificity Also the ionic bonds contribute for interactions Hydrophobic Hydrophobic Affinity H-bonds H-bonds Keep relationship between helices specificity K contact with G in major groove and phosphate backbone Mutation makes repressor affinity less than ~1000X

Cooperative DNA binding of repressor dimer 3 repressor-binding sites/operator No identical sequence Separated by 3-7 bp and AT-rich O1 has strongest affinity O1 increases the affinity of O2 Usually O3 is not filled with repressor (no enough conc.) Where to start binding 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 PRM turn/loop between helix 2 and 3 Both O1 and O2 mutations lead to virulence containing acidic patch to interact w/ basic region of RNA polymerase via electrostatic interaction -ve feedback stabilization establishment O3 O2 O1 Stabilize the RNA polymerase binding to PRM promoter site to facilitate cI transcription (transition from closed complex to open complex) Protein-protein interaction can release energy that is used to help to initiate transcription

How is the synthesis of repressor established in the first place? cI: establishment and maintenance cII: maintenance cIII: maintenance (+) via PRE promoter pN positive regulators for initiation step of cI expression (repression) initiation (cIII protects cII degraded by HflA protease) cl protein expression Inhibits translation of cro mRNA cI anti-cro RNA transcribed to cI PRE promoter needs cII protein to facilitate RNA polymerase initiating transcription

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 repressor Lambda cro molecule 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. cII regulator binds to a region extending from -25 to -45 Only when it is added, RNA polymerase can binds to PRE promoter -45 -25 -12 +13 Cis-acting mutation affects the establishment of lysogeny Similar phenotype as cII or cIII mutant (trans-acting mutants)

Lysogenic phase of λ phage The role of cII in lysogenic phase (establishment) Acts at PRE to transcribe cI Acts 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 genes via PRM and OR ， OL Block PR and PL gene expression Integration into genome All immediate early and delayed early genes are turn off

The entering lytic cycle: N: anti-termination; cro: repressor of lysogeny The role of cro repressor: dimer/9kD each Acts at OR3 to prevent RNA poly binding to PRM, blocking maintenance of cI repressor. Acts at OR/OL to prevent RNA poly from expressing immediate early genes, blocking repressor establishment. Mechanism is similar to cI repressor

A tug of war between cI and cro The 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 a helices. Two of these, a2 (blue) and a3 (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

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. Oct1 pou domain PDBcode: 1oct R = 3.0 Å R factor = 0.237 www.bioinfomaster.ulb.ac.be

Fos (mauve) – Jun (red) – Nfat (blue) PDBcode: 1a02 R = 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