NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit of RNA polymerase in Escherichia coli transcription complexes.
The effects of NusA on the RNA polymerase contacts made by nucleotides at internal positions in the nascent RNA in Escherichia coli transcription complexes were analyzed by using the photocrosslinking nucleotide analog 5-[(4-azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda PR' promoter. Crosslinks of analog in these positions in RNAs which contained either 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RNA was elongated, by placing the crosslinker nearest the 5' end of the RNA 10, 23, 24, or 44 nt away from the 3' end. The beta or beta' subunit of polymerase, and NusA when added, were contacted by RNA from 15 to 49 nt long. When the upstream crosslinker was 24 nt from the 3" end of the RNA (29-nt RNA), alpha was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to alpha. When the crosslinker was 44 nt from the 3' end (49-nt RNA), alpha crosslinks were still observed, but crosslinks to beta or beta' and NusA were greatly diminished. RNA crosslinking to alpha, and loss of this crosslink when NusA was added, was observed in the presence of NusB, NusE, and NusG and when transcription was carried out in the presence of an E. coli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha.
Liu
K
K
Department of Botany and Microbiology, University of Oklahoma, Norman 73019, USA.
Hanna
M M
MM
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000378:metabolism; D001665:Binding Sites; D003432:Cross-Linking Reagents; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D010445:Peptide Elongation Factors / Q000378:metabolism; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C070291:NusG protein, E coli; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; D012367:RNA, Viral; D012269:Ribosomal Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; C029537:ribosomal protein S10; D012321:DNA-Directed RNA Polymerases