Role of an RNase III Binding Site in Transcription Termination at λ nutL by HK022 Nun Protein

Abstract

ABSTRACT The phage HK022 Nun protein excludes phage λ by binding nascent λ p L and p R transcripts at nutL and nutR , respectively, and inducing transcription termination just downstream of these sites. Termination is more efficient at nutL than at nutR . One difference between nutL and nutR is the presence of RNase III processing sites (rIII) located immediately promoter distal to λ nutL . We found that deletion of rIII dramatically reduced Nun transcription arrest in vitro but had little effect on termination in vivo. However, consistent with the in vitro results, overexpression of a transcript carrying nutL and rIII efficiently titrated Nun, allowing λ to grow on a strain that expressed Nun, whereas a transcript carrying only nutL or nutL- rIII with nucleotides 97 to 141 deleted was ineffective. Rnc70, an RNase III mutant that binds but does not cleave rIII, also prevented Nun-mediated λ exclusion. We propose that rIII enhances the on-rate of Nun at nutL , stimulating Nun-mediated arrest in vitro. We have shown that a specific element in rIII, i.e., box C (G 89 GUGUGUG), strongly enhances arrest on rIII + templates. Nun-rIII interactions do not stimulate Nun termination in vivo, presumably because formation of the Nun- nutL complex is normally not rate-limiting in the cell. In contrast to Nun, N is not occluded by Rnc70 and is not efficiently titrated by a nutL -rIII transcript.