Active site labeling of Escherichia coli transcription elongation complexes with 5-[4-azidophenacyl)thio)uridine 5'-triphosphate.
Escherichia coli RNA polymerase transcription elongation complexes have been prepared that contain a photo-cross-linking uridine analog at only the 3' end, or one or two nucleotides removed from the 3' end, in the nascent RNA chain. Additionally, complexes have been isolated in which the analog has been substituted for every UMP residue, at positions ranging from 20 to 140 nucleotides from the 3' end. The RNA has been photochemically cross-linked to the RNA polymerase to identify the subunits that form the binding site(s) for these regions in the nascent RNA. The photo-cross-linking nucleotide analog used for these studies was 5-[4-azidophenacyl)thio)uridine-5'-triphosphate (5-APAS-UTP), which acts as a 10-15 A probe. With 5-APAS-UMP positioned only at the 3' end of the RNA, or one or two nucleotides from the 3' end, only the beta subunit appeared to be contacted. When the analog was positioned throughout the RNA, both the beta and beta' subunits were contacted. No labeling of the sigma or alpha subunits was observed with any RNA length. In addition to placing this analog at specific positions in short RNAs, we have carried out transcription studies with 5-APAS-UTP to determine the optimal UTP to analog ratio for production of full length, photoreactive transcripts. Surprisingly, we found that when transcription complexes were stalled shortly after initiation, by deletion of one ribonucleoside triphosphate to synchronize transcription, changes in transcriptional pausing occurred downstream. These results suggest that events that occur early in transcription can affect the elongation and/or termination events that occur far downstream from the promoter. This effect occurred even with UTP but was greatly enhanced by replacement of UTP with either this analog or 4-thio-UTP. By enhancing the normal transcriptional pausing event, these analogs can serve as probes of the conformational changes that may exist in paused transcription complexes.
Dissinger
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Department of Biological Chemistry, California College of Medicine, University of California, Irvine 92717.
Hanna
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D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D012329:RNA, Bacterial; C061065:5-((4-azidophenacyl)thio)uridine 5'-triphosphate; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate