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

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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.