The NusA N-terminal domain is necessary and sufficient for enhancement of transcriptional pausing via interaction with the RNA exit channel of RNA polymerase.
NusA is a core, multidomain regulator of transcript elongation in bacteria and archaea. Bacterial NusA interacts with elongating complexes and the nascent RNA transcript in ways that stimulate pausing and termination but that can be switched to antipausing and antitermination by other accessory proteins. This regulatory complexity of NusA likely depends on its multidomain structure, but it remains unclear which NusA domains possess which regulatory activity and how they interact with elongating RNA polymerase. We used a series of truncated NusA proteins to measure the effect of the NusA domains on transcriptional pausing and termination. We find that the N-terminal domain (NTD) of NusA is necessary and sufficient for enhancement of transcriptional pausing and that the other NusA domains contribute to NusA binding to elongating complexes. Stimulation of intrinsic termination requires higher concentrations of NusA and involves both the NTD and other NusA domains. Using a tethered chemical protease in addition to protein-RNA cross-linking, we show that the NusA NTD contacts the RNA exit channel of RNA polymerase. Finally, we report evidence that the NusA NTD recognizes duplex RNA in the RNA exit channel.
Ha
Kook Sun
KS
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Toulokhonov
Innokenti
I
Vassylyev
Dmitry G
DG
Landick
Robert
R
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*; D035602:Transcriptional Elongation Factors
D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D012321:DNA-Directed RNA Polymerases