NUSA INTERFERES WITH INTERACTIONS BETWEEN THE NASCENT RNA AND THE C-TERMINAL DOMAIN OF THE A 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 transcri ption complexes were analyzed by using the photocrosslinking nucleotid e analog 5-[(4-azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda P-R, promo ter. Crosslinks of analog in these positions in RNAs which contained e ither 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RN A 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' subun it of polymerase, and NusA when added, were contacted by RNA from 15 t o 49 nt long. When the upstream crosslinker was 24 nt from the 3 '' en d of the RNA (29-nt RNA), alpha was also contacted in the absence of N usA. The addition of NusA prevented RNA crosslinking to alpha. When th e 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 gre atly 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. c oli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha.