THE RIBOSOMAL ENVIRONMENT OF TRANSFER-RNA - CROSS-LINKS TO RIBOSOMAL-RNA FROM POSITION-8 AND POSITION-20 1 IN THE CENTRAL FOLD OF TRANSFER-RNA LOCATED AT THE A-SITE, P-SITE, OR E-SITE/

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridin e (''acp3U'') at position 20:1 of lupin tRNA(Met) was coupled to a pho toreactive diazirine derivative. Similarly, the 4-thiouridine at posit ion 8 of Escherichia coli tRNA(Phe) was modified with an aromatic azid e. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for th e A, P, or E sites. After photoactivation of the diazirine or azide gr oups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribon uclease H digestion and primer extension analysis. The crosslinked rib osomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink f rom tRNA position 20:1 to the loop-end region (nt 877-983) of helix 38 of the 23S RNA (a region that has not so far been associated at all w ith tRNA binding), and (2) a largely P-site-specific crosslink from tR NA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belong ing to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central f old of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).