SYNTHESIS AND CHARACTERIZATION OF A NEW PHOTO-CROSS-LINKING CTP ANALOG AND ITS USE IN PHOTOAFFINITY-LABELING ESCHERICHIA-COLI AND T7-RNA POLYMERASES

A new photocrosslinking CTP analog that functioned as a substrate duri ng transcription was synthesized and used to photoaffinity label E.col i and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophen-a cyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 angstrom from the nucleotide base and specific ally replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases in efficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of 0-modification of E.coli RNA polymerase, downstream of the lambda PR' promoter (+ 16), a pause that requires specific DNA sequences but no apparent RNA hairp in. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier r esults involving a NusA-enhanced pause site downstream from an RNA hai rpin.