Use of reverse transcription to determine the exact locations of psoralen photochemical crosslinks in RNA.

We have studied the properties of avian myeloblastosis virus reverse transcriptase on Escherichia coli 16S ribosomal RNA that was known to contain a psoralen crosslink in a restricted region around residue 920. These crosslinked RNA molecules were purified on the basis of loop size by gel electrophoresis. Reverse transcription stopped at specific positions in the crosslinked molecules but not in control linear molecules. With the particular crosslink that was studied, at the earliest time of reverse transcription, the most frequent stopping site was G925. At later times two nearly equal stops at G925 and C924 were seen. The crosslinked site was an absolute stop since even at long times of incubation the reverse transcriptase was not able to proceed beyond G925/C924. An independent determination of the crosslinked site by directly sequencing a section of the crosslinked RNA indicated that C924 was the sole nucleotide involved in the crosslink. Therefore, reverse transcriptase was able to synthesize cDNA all the way up to and including the nucleotide that contained the psoralen crosslink. Thus, reverse transcription can be a rapid and precise method for determining psoralen crosslinking sites when prefractionated, crosslinked RNA templates are used.