THE DECODING REGION OF 16S RNA - A CROSS-LINKING STUDY OF THE RIBOSOMAL A-SITE, P-SITE AND E-SITE USING TRANSFER-RNA DERIVATIZED AT POSITION-32 IN THE ANTICODON LOOP

A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.