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

Abstract

To test the potential of psoralen photoaddition for the probing of RNA conformation at sequence resolution, the specificity of the reaction of 4''-(hydroxymethyl)-4,5'',8-trimethylpsoralen (HMT) with E. coli tRNAPhe was analyzed. The sites of HMT covalent addition were identified by a combination of analytical techniques involving chemical cleavage of the tRNAPhe molecule at the m7G [7-methylguanine] site and gel electrophoresis of RNase T1 digests together with paper electrophoretic characterization of HMT-modified nucleotides and oligonucleotides. Advantage was taken of the alteration of the cleavage rate of pancreatic RNase adjacent to a photoadduct. HMT photoaddition showed a very high preference for uracil residues. Important differences in HMT photoreactivity were observed for various U sites of the tRNAPhe molecule. Reactivity of specific bases was correlated with partial melting of the molecule. There appears to be 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.