Two-dimensional gel electrophoresis technique for determination of the cross-linked nucleotides in cleavable covalent RNA-RNA complexes. Application to Escherichia coli and Bacillus subtilis acetylvalyl-tRNA covalently linked to E. coli 16S and yeast 18S ribosomal RNA.

We have developed a new method which yields in a single step the site of cross-linking between two oligonucleotides covalently linked by a cleavable bond. The isolated duplex, labeled at both 5'-ends, is split randomly and then analyzed by diagonal gel electrophoresis with cleavage of the cross-link between the two gel dimensions. Digestion products which do not contain the cross-link migrate along the diagonal, while products resulting from cleavage of the cross-link migrate as off-diagonal products. The site of cross-linking is determined by analysis of both diagonal and off-diagonal products. This method was successfully applied to three different oligonucleotide duplexes isolated by T1 RNase digestion from Escherichia coli tRNA covalently linked at the ribosomal P site to either Escherichia coli 16S RNA or yeast 18S RNA and from Bacillus subtilis tRNA cross-linked to Escherichia coli 16S RNA. The site of cross-linking was unambiguously localized to C1400 in Escherichia coli 16S RNA and to the equivalent position, C1626, in yeast 18S RNA. Direct evidence was also provided for the participation of the wobble base (c)mo5U34 of the tRNA in the cross-link. Furthermore, our results exclude the possibility of minor cross-linking sites at other positions. This new method is reliable, rapid, and easy to handle and should be applicable to any cleavable covalent RNA-RNA duplex. Furthermore, it is sensitive to certain aspects of the steric conformation of such covalent duplexes.