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-thiocytidi ne residue at position 32 of tRNA(Arg)I from Escherichia coli. This mo dified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, th e sites of cross-linking to 16S rRNA were identified by our standard p rocedures. Each of the three tRNA binding sites showed a characteristi c pattern of cross-linking. From tRNA at the A site, a major cross-lin k was observed to position 1378 of the 16S RNA, and a minor one to pos ition 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 1 338. The E site bound tRNA showed major cross-links to position 693 (i dentical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immun ological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all thre e tRNA sites, with S11 as a minor product. The results are discussed i n terms of the overall topography of the decoding region of the 30S ri bosomal subunit.