Efficient inhibition of Escherichia coli RNA polymerase by the bacteriophage T4 AsiA protein requires that AsiA binds first to free sigma(70)

The bacteriophage T4 AsiA protein inhibits transcription from host and phag e early promoters and is required, along with the T4 MotA protein, for acti vation of phage middle promoters. During infection, AsiA is found in a tigh t association with the sigma (70) subunit of RNA polymerase. We show that A siA binds rapidly to free sigma (70) at either 4 degreesC or 30 degreesC to form an AsiA-sigma (70) complex that with core efficiently reconstitutes t he AsiA-inhibited RNA polymerase. In contrast, AsiA does not inhibit transc ription after a 15 minute incubation with RNA polymerase holoenzyme at 4 de greesC, and at 30 degreesC 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 w ith the momentary dissociation of holoenzyme to give free sigma (70) and co re. Our results indicate that AsiA is either unable to access holoenzyme di rectly or does so very slowly. Efficient generation of the AsiA-inhibited R NA polymerase requires that AsiA first binds to free sigma (70) and then th e AsiA-sigma (70) complex binds to core to form the AsiA-inhibited polymera se.