Role of E.coli NusA in phage HK022 Nun-mediated transcription termination.
The 109 amino acid residue Nun protein expressed from prophage HK022 excludes superinfecting phage lambda by arresting transcription on the lambda chromosome near the lambdanut sites. In vitro, the Nun N terminus binds to nascent lambdanutRNA, whereas the C terminus interacts with RNA polymerase and DNA template. Escherichia coli host factors, NusA, NusB, NusE (S10), and NusG, stimulate Nun-arrest. NusA binds the Nun C terminus and enhances formation of the Nun-nutRNA complex. Because of these in vitro activities of NusA, and since a nusA mutation (nusAE136K) blocked Nun in vivo, we assumed that NusA was required for Nun activity. However, using a nusAts strain, we find that NusA is required for termination at nutR but not at nutL. Furthermore, nusAE136K is dominant to nusA(+) for Nun-arrest, both in vitro and in vivo. NusAE136K shows increased affinity for Nun and, unlike NusA(+), can readily be recovered in a ternary complex with Nun and nutRNA. We propose NusAE136K suppresses Nun-arrest when it is a component of the transcription elongation complex, perhaps, in part, by blocking interactions between the Nun C terminus and RNA polymerase and DNA. We also find that in contrast to Nun-arrest, antitermination by lambda N requires NusA.
Kim
Hyeong C
HC
Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY 10032, USA.
Washburn
Robert S
RS
Gottesman
Max E
ME
D025523:Bacteriophage HK022 / Q000235:genetics / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008957:Models, Genetic; D009154:Mutation; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C073501:Nun protein, Enterobacteria phage HK022; D010445:Peptide Elongation Factors; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D014764:Viral Proteins; C068727:nusA protein, E coli; D012321:DNA-Directed RNA Polymerases