Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA

Abstract

An aryl trifluoromethyl diazirine photoreactive derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(IArg) and this derivatized tRNA was bound to Escherichia coli 70S ribosomes. After irradiation at 350 nm the site of cross-linking to the 16S RNA was analyzed by our standard procedures and found to lie within the secondary structural element comprising bases 956-983; this region contains two modified nucleotides at positions 966 and 967. Similarly, an aryl azido photoreactive derivative was attached to the phenylalanine residue of Phe-tRNA(Phe), and the derivatized aminoacyl tRNA was bound to the ribosome either at the A- or the P-site. In both cases, after irradiation at 250 nm, the cross-link site was localized to position 2439 of the 23S RNA; in the secondary structure of the latter the neighboring nucleotide 2442 is base-paired to a modified nucleotide at position 2069. Taken together with other cross-linking data, these results now directly implicate a total of 27 out of the 29 modified nucleotides in E. coli 16S and 23S RNA as lying within or close to the functional center of the ribosome.