The Mechanism of Transcriptional Activation by the Topologically DNA-linked Sliding Clamp of Bacteriophage T4

Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the σ-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 (∼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 °C, but once opened at 30 °C remains open upon shift to the lower temperature. At a hybrid promoter for σ 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.