RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits: a method for the direct analysis of the RNA regions involved in the cross-links

Abstract

A prerequisite for topographical studies on ribosomal sub-units involving RNA-protein cross-linking is that the cross-linking sites on the RNA should be determined. Methodology is presented which offers a solution to this problem, using as a test system 30S subunits in which protein S7 has been cross-linked to the 16S RNA by ultraviolet irradiation. The method is based on a gel separation system in the presence of a non-ionic detergent. When a ribonucleoprotein fragment containing RNA-protein cross-links is applied to this system, non-cross-linked protein is removed, and simultaneously the cross-linked RNA-protein complex is separated from non-cross-linked RNA. Oligo-nucleotide analysis of the S7-RNA complex isolated in this manner showed it to consist of a region of RNA from sections P-A of the 16S RNA. A single characteristic oligonucleotide was absent from this region, and it was tentatively concluded that this missing oligonucleotide contains the actual site of cross-linking.