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 at 302 or 337 pm, Transcription complexes containing RNA substituted with 5-SH-UMP were post-transcriptionally modified to attach a photocrosslinking group to thiol-tagged nucleotides in the RNA on the surface of the polymerase or free in solution. The pK(a) 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.