Identification of sites of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen cross-linking in Escherichia coli 23S ribosomal ribonucleic acid.
The reagent 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) was used to cross-link 23S rRNA from Escherichia coli under 50S ribosomal subunit reconstitution conditions. Following partial digestion of the RNA with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate fragments derived from the cross-linked sites. These fragments were analyzed by digestion with ribonucleases T1 and A and their positions in the 23S RNA sequence identified. Fragment a1 (positions 1325-1426) is cross-linked to a2 (positions 1574-1623); fragment b1 (positions 1700-1731) is cross-linked to b2 (positions 1732-1753); and a cross-link is formed within fragment c (or c') (positions 863-916). In the latter case, the cross-link was located precisely, linking residues C867 and U913. All three HMT-mediated cross-links are consistent with a proposed secondary structure model for 23S RNA [Noller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, R. R., Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A., Gupta, R., & Woese, C. R. (1981) Nucleic Acids Res. 9, 6167-6189].
D001483:Base Sequence; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism*; D011564:Furocoumarins; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012335:RNA, Ribosomal / Q000378:metabolism*; D014307:Trioxsalen / Q000031:analogs & derivatives
D003432:Cross-Linking Reagents; D011564:Furocoumarins; D012335:RNA, Ribosomal; C016507:hydroxymethyltrioxsalen; D014307:Trioxsalen