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Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the sigma-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer-template junctions by its clamp loader. The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes. The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly. Activation ( approximately 320-fold in this work) increases affinity in the closed complex and accelerates promoter opening. Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 degrees C, but once opened at 30 degrees C remains open upon shift to the lower temperature. At a hybrid promoter for sigma(70) and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening. Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33.

In bacterial transcription, diverse sigma-family promoter recognition proteins compete for a common RNA polymerase core. Bacteriophage T4 infection ultimately reduces this competition to a duel between activated viral middle and enhanced late transcription, involving two sigma proteins, two phage-encoded activator proteins and two phage-specific co-activators. This competition has been analyzed in vitro, and the relative abundances in T4-infected Escherichia coli of the participating proteins have been measured. Activated late transcription holds an advantage over activated middle transcription, especially at higher ionic strength. This advantage is further compounded by ADP-ribosylation of the RNA polymerase alpha subunits, and by the phage-specific, RNA polymerase core-bound RpbA subunit. The largest contribution to the middle-late competition is made by gp55, the late sigma factor, but not enough of gp55 is produced during T4 infection to shut off middle transcription by direct competition with sigma(70). AsiA, the originally identified anti-sigma protein is not a major determinant of middle-late competition.