Sequencing psoralen photochemically reactive sites in Escherichia coli 16 S rRNA.

The photochemical modification of 16 S rRNA by longwave ultraviolet light in the presence of hydroxymethyltrimethyl psoralen (HMT) produces uridine-psoralen adducts. Psoralenmodified 16 S rRNA is primed with a 3’-proximal restriction fragment and copied with reverse transcriptase. The average length of the cDNA transcripts decreases as the number of covalently bound psoralen adducts on the template RNA increases. Adducts stop reverse transcriptase and abbreviated cDNAs terminate at the positions of the lesions. The cDNA reverse transcripts of psoralen-reacted 16 S rRNA are electrophoresed in parallel with a dideoxy sequence (Sanger method) which uses the same restriction fragment as a primer. The psoraleninduced cDNA stops are found to be highly localized hot spots. In the 1 SO-nucleotide sequence investigated at sequence resolution, two hot spots for psoralen reaction are found. In both cases the psoralen-induced stops are at UpU sequences near the bases of predicted hairpins.