Coliphage HK022 Nun protein inhibits RNA polymerase translocation.
The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β' subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5' edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.
Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032; and.
Christal L
CL
Vitiello
Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, Center for Cancer Research, Frederick, MD 21702.
Maria L
ML
Kireeva
Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, Center for Cancer Research, Frederick, MD 21702.
Lucyna
L
Lubkowska
Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, Center for Cancer Research, Frederick, MD 21702 kashlevm@mail.nih.gov meg8@columbia.edu.
Mikhail
M
Kashlev
Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032; and kashlevm@mail.nih.gov meg8@columbia.edu.
Max
M
Gottesman
D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D013698:Templates, Genetic; D061805:Transcription Elongation, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D011756:Diphosphates; D009711:Nucleotides; C073501:Nun protein, Enterobacteria phage HK022; D012367:RNA, Viral; D014157:Transcription Factors; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases
Bacteriophage HK022; Bacteriophage Lambda; Exclusion; Transcription Elongation; Transcription Termination