Identification of sites of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen crosslinking in Escherichia coli 23S ribosomal ribonucleic acid

Publisher Website
Google Scholar
Abstract
The reagent 4''-(hydroxymethyl)-4,5'',8-trimethylpsoralen (HMT) was used to cross-link 23S rRNA from E. coli under 50S ribosomal subunit reconstitution conditions. Following partial digestion of the RNA with RNase T1, 2-dimensional diagnoal electrophoresis in denaturing polyacrylamide gels was used to isolate fragments derived from the cross-linked sites. These fragments were analyzed by digestion with RNases 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 3 HMT-mediated cross-links are consistent with a proposed secondary structure model for 23S RNA.