Dissection of the bacteriophage T4 late promoter complex.
Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator is gp45, the viral replisome's sliding clamp; two sliding-clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) sigma subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The C-terminal sliding-clamp-binding epitopes of gp33 and gp55 are perfectly interchangeable, but the functions of these two RNAP-sliding clamp connections differ: only the gp33-gp45 linkage is essential for activation, while loss of the gp55-gp45 linkage impairs but does not abolish activation. Formation of transcription-ready promoter complexes by the sliding-clamp-activated wild-type T4 RNAP resists competition by high concentrations of the polyanion heparin. This avid formation of promoter complexes requires both linkages of the T4 late RNAP to the sliding clamp. Preopening the promoter compensates for loss of the gp55-gp45 but not the gp33-gp45 linkage. We interpret the relationship of these findings and our prior analysis to the common model of transcriptional initiation in bacteria in terms of two parallel pathways, with two RNAP holoenzymes and two DNA templates: (1) gp55-RNAP and the T4 late promoter execute basal transcription; (2) gp55-gp33-RNAP and the T4 late promoter with its mobile enhancer, the T4 sliding clamp, execute activated transcription. gp55 and gp33 perform sigma-like functions, gp55 in promoter recognition and gp33 (as well as gp55) in enhancer recognition. gp33 operates the switch between these two pathways by repressing basal transcription.
Nechaev
Sergei
S
Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA. nechaevs@niehs.nih.gov
Geiduschek
E Peter
EP
D017122:Bacteriophage T4 / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D046911:Macromolecular Substances / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit