PHOTO-CROSS-LINKING ANALYSIS OF PROTEIN-RNA INTERACTIONS IN ESCHERICHIA-COLI TRANSCRIPTION COMPLEXES

Regulation of transcription involves numerous specific protein-nucleic acid interactions. We have utilized photochemical crosslinking to ide ntify interactions between Escherichia coli transcription proteins and the nascent RNA in several transcription complexes, including initiat ion, elongation, and antitermination complexes. We have developed new nucleotide analogs, 5-APAS-UTP and 5-APAS-CTP, which are tagged with p hotocrosslinking groups on base positions that do not interfere with n ormal Watson-Crick base-pairing. These analogs are incorporated at int ernal positions in RNA by E. coli RNA polymerase without disrupting RN A secondary structures. We have also used 8-azido-ATP, which can be in corporated uniquely into the 3' end of the RNA, to analyze interaction s at the enzyme active site. Interactions between the RNA and the poly merase subunits, and the effect of various transcription factors, incl uding NusA, NusB, NusE, and NusG, have been examined in complexes cont aining RNAs from 4 to approximately 80 nucleotides. At almost every RN A position examined, both the beta and beta' subunits are contacted, b ut never the a subunit or NusA. An effect of NusA on the core labeling has been observed in some complexes, however. Sigma is contacted by n ucleotides within three nucleotides of the +1 position on the DNA.