Features of the a T4 virion Over 30 proteins; complex tail (tube, sheath, whiskers, base plate, pins, tail fibers). During a T4 penetration, it's virion uses this notable feature It has ATP-dependent tail contraction, which punctures the cell and injects the viral contents. A T4 penetration causes these substantial injuries Large hole in cell wall and membrane; depolarizes membrane; cell contents flow out; initiates emergency response in host where host expression ceases while injury is repaired; Lysis-from-without can occur at high MOI. T4 does this during intermediate-early expression Convert cell to viral purpose; Use cellular RNA Pol, sigma-70, rho. T4's intermediate-early products Prepare cell for viral multiplication: superinfection protection; RNA pol modifying enzyme (ADP-ribosylation); Nucleases to digest host chromosome. Superinfection protection Alters the cell membrane so other virions cannot attach. T4 uses this to modify RNA Pol which in turn does this ADP-ribosylation; Recognizes new set of promoters. T4 has this feature of its DNA The C's are Hydroxymethyl Cytosines. This is required so that the T4 nucleases digest the host chromosome and not the new phage DNA. T4 does this during delayed-early expression DNA replication, using a modified RNA Pol (one subunit ADP-ribosylated, recognizes new set of promoters). Produces replication enzymes: Cytosine-modifying protein, another RNA pol modifying protein. T4 does this in intermediate gene expression DNA replication, and shift to late expression. Uses doubly modified RNA Pol, and new promoters. Produces replication enzymes: new RNA pol subunit (to replace sigma), strand switch protein. T4 does this during late gene expression Produce assembly products; Uses RNA Pol with phage subunits; only transcribes replicated DNA; transcribes on late strand (a different strand is used for early, intermediate). gp45 T4's sliding clamp protein gp33 An activator used by T4 gp55 T4's replacement sigma factor used during late expression Subunits that make up RNA Pol's core complex alpha, alpha, beta, beta prime, sigma The problem with replicating T4's linear dsDNA, and how it addresses it Progeny strands have primers at their 5' ends, and no telomerase activity is available. T4 solves this using Circular Permutation and Terminal Redundancy. Circular Permutation When linear chromosomes show genetic linkage maps characteristic of circular chromosomes. These are created using long linear concatamers, e.g., ABCDEFGHABCDEFGHABCDEFGHABCDEFGHABC. Therefore, only the ends of the concatamer has the primers left on, but these are internalized where they can be removed by Pol I. Main results from Kolesky experiment gp55 is limiting for direct competition between the T4 middle and late gene-transcribing holoenzymes; AsiA neither stimulates nor inhibits T4 late transcription and does not give a competitive advantage to late promoters over middle promoters. Kolesky showed that two factors influence competition Strength of the promoter; action of DNA binding proteins (can be positive or negative). Presence of MotA implies this Host's sigma70 is no longer able to recognize -35 MotA T4 uses this DNA binding protein for the -35 region AsiA Protein that binds RNA Pol, so it recognizes MotA which is bound at -35. Transcribes in T4 early expression RNA Pol and host sigma70 Transcribes in T4 middle expression Modified RNA Pol and host sigma70. Regulated by MotA and AsiA. Transcribes in T4 late expression Modified RNA Pol, gp55 (instead of host sigma70). Also requires gp33 (activator) and gp45 (sliding clamp). Why does the Kolesky experiment have an overhang on one of the strands? This is needed because gp45 needs to load on a single strand region. In the Kolesky experiment, what is the purpose of the EcoRI-E111Q DNA-binding protein? It prevents the sliding clamp (gp45) from sliding past the end of the strand and falling off. For T4 middle expression, what is present? sigma70, MotA, AsiA, RNA Pol For T4 late expression, what is present? gp55, gp45, gp33, RNA Pol Since there is less gp55 than sigma70 present, this implies that... Both middle and late expression are taking place and are in competition with each another.