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.
Hanna
M M
MM
Department of Botany-Microbiology, University of Oklahoma, Norman 73019.
D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors; D011485:Protein Binding; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012808:Sigma Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism
D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; D012808:Sigma Factor; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; C061065:5-((4-azidophenacyl)thio)uridine 5'-triphosphate; C081396:5-((4-azidophenacyl)thio)cytidine-5'-triphosphate; D003570:Cytidine Triphosphate; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate