Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E. coli: a model for the tertiary structure of the tRNA binding domain in 23S RNA

Abstract

Intramolecular RNA cross-links were induced within the large ribosomal subunit of E. coli by mild ultraviolet irradiation. Regions of the 23S RNA previously implicated in interactions with ribosomal-bound tRNA were then specifically excised by addressed cleavage using ribonuclease H, in conjunction with synthetic complementary decadeoxyribonucleotides. Individual cross-linked fragments within these regions released by such 'directed digests' were isolated by two-dimensional gel electrophoresis and the sites involved in the cross-links determined using classical oligonucleotide analysis techniques. Using this approach, seven 'new' cross-links could be precisely localised, between positions 1782 and 2608-2609, 1940 and 2554, 1941-1942 and 1964-1965, 1955 and 2552-2553, 2145-2146 and 2202, 2518-2519 and 2544-2545, and between positions 2790-2791 and 2892-2895 in the 23S RNA sequence. These data, in conjunction with data from RNA-protein cross-linking studies carried out in our laboratory, were used to define a model for the tertiary organisation of the tRNA binding domain of 23S RNA 'in situ', in which the specific nucleotides associated with tRNA binding in the 'A' and 'P' sites are clustered at the base of the 'central protuberance' of the 50S subunit.