Structural basis of transcription arrest by coliphage HK022 Nun in anEscherichia coliRNA polymerase elongation complex.
Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures ofEscherichia coliRNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β' subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.
Laboratory of Molecular Biophysics, The Rockefeller University, New York City, United States.
Jin Young
JY
Kang
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York City, United States.
Paul Dominic B
PD
Olinares
Laboratory of Molecular Biophysics, The Rockefeller University, New York City, United States.
James
J
Chen
Laboratory of Molecular Biophysics, The Rockefeller University, New York City, United States.
Elizabeth A
EA
Campbell
Public Health Research Institute, Newark, United States.|Department of Microbiology and Molecular Genetics, Rutgers Biomedical and Health Sciences, Newark, United States.|Rutgers New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, United States.
Arkady
A
Mustaev
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York City, United States.
Brian T
BT
Chait
Department of Microbiology and Immunology, Columbia University Medical Center, New York City, United States.
Max E
ME
Gottesman
Laboratory of Molecular Biophysics, The Rockefeller University, New York City, United States.
Seth A
0000-0002-8241-3153
SA
Darst
D020285:Cryoelectron Microscopy; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology / Q000821:virology*; D011485:Protein Binding; D011487:Protein Conformation; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
C073501:Nun protein, Enterobacteria phage HK022; D014157:Transcription Factors; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases
Cryo-electron microscopy; E. coli; HK022; Nun; RNA polymerase; Transcription; biochemistry; biophysics; structural biology