Specificity of the photoreaction of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen with RNA. Identification of reactive sites in E. coli tRNAPhe

Abstract

In order to test the potential of psoralen photoaddition for the probing of RNA conformation at sequence resolution, we have analyzed the specificity of the reaction of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) with Escherichia coli tRNAPhe. The sites of HMT covalent addition have been identified by a combination of analytical techniques involving chemical cleavage of the tRNAPhe molecule at the m7G site and gel electrophoresis of RNase T1 digests together with paper electrophoretic characterization of HMT-modified nucleotides and oligonucleotides. In addition, we have taken advantage of the alteration of the cleavage rate of pancreatic RNase adjacent to a photoadduct. HMT photoaddition shows a very high preference for uracil residues. However, important differences in HMT photoreactivity are observed for various U sites of the tRNAPhe molecule. Reactivity of specific bases has been correlated with partial melting of the molecule. Data available so far indicate a strong preference of the photoreactive probe for a "loose" helical conformation as compared with a tight helix, whereas a random coil appears poorly reactive.