Transcription termination by phage HK022 Nun is facilitated by COOH-terminal lysine residues.
The 109-amino acid Nun protein of prophage HK022 excludes superinfecting bacteriophage lambda by blocking transcription elongation on the lambda chromosome. Multiple interactions between Nun and the transcription elongation complex are involved in this reaction. The Nun NH(2)-terminal arginine-rich motif binds BOXB sequence in nascent lambda transcripts, whereas the COOH terminus binds RNA polymerase and contacts DNA template. Nun Trp(108) is required for interaction with DNA and transcription arrest. We analyzed the role of the adjacent Lys(106) and Lys(107) residues in the Nun reaction. Substitution of the lysine residues with arginine (K106R/K107R) had no effect on transcription arrest in vitro or in vivo. Nun K106A/K107A was partially active, whereas Nun K106D/K107D was defective in vitro and failed to exclude lambda. All mutants bound RNA polymerase and BOXB. In contrast to Nun K106R/K107R and K106A/K107A, Nun K106D/K107D did not cross-link DNA template. These results suggest that transcription arrest is facilitated by electrostatic interactions between positively charged Nun residues Lys(106) and Lys(107) and negatively charged DNA phosphate groups. These may assist intercalation of Trp(108) into template.
Kim
Hyeong C
HC
Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, New York 10032, USA.
Gottesman
Max E
ME
D000595:Amino Acid Sequence; D025523:Bacteriophage HK022 / Q000235:genetics*; D001667:Binding, Competitive; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D066298:In Vitro Techniques; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010710:Phosphates / Q000737:chemistry; D055672:Static Electricity; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
C073501:Nun protein, Enterobacteria phage HK022; D010710:Phosphates; D014157:Transcription Factors; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; D008239:Lysine