Preparation of probe-modified RNA with 5-mercapto-UTP for analysis of protein-RNA interactions.
We report a modified synthesis for 5-mercapto-UTP (5-SH-UTP) and its use for analysis of protein-RNA interactions utilizing Escherichia coli and T7 RNA polymerases and yeast RNA polymerases I and III. 5-SH-UTP did not affect transcriptional pausing, Rho-independent termination or recognition of the E. coli transcription complex by NusA. RNA containing 5-SH-UMP did not crosslink to polymerase when irradiation was 302 or 337 nm. Transcription complexes containing RNA substituted with 5-SH-UMP were post-transcriptionally modified to attach a photocross-linking group to thiol-tagged nucleotides in the RNA on the surface of the polymerase of free in solution. The pKa for 5-SH-UTP was determined to be 5.6, so modification of the thiol groups in the RNA with p-azidophenacyl bromide could be carried out at pH 7. Addition of the transcription termination factor Rho, a RNA binding protein, to E. coli transcription complexes resulted in RNA crosslinking to Rho and to the beta and beta' subunits of polymerase. Using 5-SH-UTP, one can distinguish RNA binding domains on the surface of RNA polymerases or other RNA binding proteins from those buried within the protein.
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Department of Botany/Microbiology, University of Oklahoma, Norman 73019, USA.
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Hanna
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D001483:Base Sequence; D003432:Cross-Linking Reagents; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010777:Photochemistry; D011485:Protein Binding; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D015347:RNA Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000138:chemical synthesis; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis
C093399:5-mercaptouridine triphosphate; D003432:Cross-Linking Reagents; D015347:RNA Probes; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D013873:Thionucleotides; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate