NusA interferes with interactions between the nascent RNA and the C-terminal domain of the α subunit of RNA polymerase in Escherichia coli transcription complexes

The effects of NusA on the RNA polymerase ontacts 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 λP_R′ 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 β or β′ 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), α was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to α. When the crosslinker was 44 nt from the 3′ end (49-nt RNA), α crosslinks were still observed, but crosslinks to β or β′ and NusA were greatly diminished. RNA crosslinking to α, 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 α.