Specificity of the photoreaction of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen with ribonucleic acid. Identification of reactive sites in Escherichia coli phenylalanine-accepting transfer ribonucleic acid

• Published in: Biochemistry (Easton) 1982-03, Vol.21 (6), p.1357-1363
• Main Authors: Bachellerie, J P, Hearst, J E
• Format: Article
• Language: English
• Subjects: Binding Sites; Escherichia coli - metabolism; Furocoumarins - metabolism; Nucleic Acid Conformation; Photochemistry; RNA, Bacterial - metabolism; RNA, Transfer, Amino Acyl - metabolism; Trioxsalen - analogs & derivatives; Trioxsalen - metabolism
• ISSN: 0006-2960
• DOI: 10.1021/bi00535a039

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.