Efficient inhibition of Escherichia coli RNA polymerase by the bacteriophage T4 AsiA protein requires that AsiA binds first to free sigma70.
The bacteriophage T4 AsiA protein inhibits transcription from host and phage early promoters and is required, along with the T4 MotA protein, for activation of phage middle promoters. During infection, AsiA is found in a tight association with the sigma70 subunit of RNA polymerase. We show that AsiA binds rapidly to free sigma70 at either 4 degrees C or 30 degrees C to form an AsiA-sigma70 complex that with core efficiently reconstitutes the AsiA-inhibited RNA polymerase. In contrast, AsiA does not inhibit transcription after a 15 minute incubation with RNA polymerase holoenzyme at 4 degrees C, and at 30 degrees C an incubation of several minutes is required to inhibit most of the polymerase. We show that the heat step needed for AsiA is not the formation of an active AsiA protein. However, it is consistent with the momentary dissociation of holoenzyme to give free sigma70 and core. Our results indicate that AsiA is either unable to access holoenzyme directly or does so very slowly. Efficient generation of the AsiA-inhibited RNA polymerase requires that AsiA first binds to free sigma70 and then the AsiA-sigma70 complex binds to core to form the Asi-A-inhibited polymerase.
Hinton
D M
DM
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830, USA. dhinton@helix.nih.gov
Vuthoori
S
S
D001051:Apoenzymes / Q000378:metabolism; D017122:Bacteriophage T4 / Q000737:chemistry*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D020035:Holoenzymes / Q000378:metabolism; D006358:Hot Temperature; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits; D012808:Sigma Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D001051:Apoenzymes; C078876:AsiA protein, Enterobacteria phage T4; D020035:Holoenzymes; D021122:Protein Subunits; D012808:Sigma Factor; D014764:Viral Proteins; D004247:DNA; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases