Photocrosslinking analysis of protein-RNA interactions in E. coli transcription complexes

Regulation of transcription involves numerous specific protein-nucleic acid interactions. We have utilized photochemical crosslinking to identify interactions between Escherichia coli transcription proteins and the nascent RNA in several transcription complexes, including initiation, elongation, and antitermination complexes. We have developed new nucleotide analogs, 5-APAS-UTP and 5-APAS-CTP, which are tagged with photocrosslinking groups on base positions that do not interfere with normal Watson-Crick base-pairing. These analogs are incorporated at internal positions in RNA by E. coli RNA polymerase without disrupting RNA secondary structures. We have also used 8-azido-ATP, which can be incorporated uniquely into the 3' end of the RNA, to analyze interactions at the enzyme active site. Interactions between the RNA and the polymerase subunits, and the effect of various transcription factors, including NusA, NusB, NusE, and NusG, have been examined in complexes containing RNAs from 4 to approximately 80 nucleotides. At almost every RNA position examined, both the beta and beta' subunits are contacted, but never the alpha subunit or NusA. An effect of NusA on the core labeling has been observed in some complexes, however. Sigma is contacted by nucleotides within three nucleotides of the +1 position on the DNA.