Crosslinking of the anticodon of P and A site-bound tRNAs to the ribosome via aromatic azides of variable length: involvement of 16S rRNA at the A site

Abstract

The topography of the ribosomal decoding site was explored by affinity labeling from the 5'-anticodon base, 5-(carboxymethoxy)uridine-34, of P or A site bound tRNA1Val. A nitrophenyl azide was attached to the carboxyl group of this nucleotide via side chains varying in length from 18 to 24 A. Binding of acetylvalyl-tRNA to the P site was codon dependent and that of valyl-tRNA to the A site was both codon and elongation factor Tu (EFTu) dependent. Cross-linking to both A and P sites was irradiation, probe, codon, and, in the case of the A site, EFTu dependent. Putative P-site cross-linked aminoacyl-tRNA was reactive with puromycin. The yield of cross-linking was little affected by placement of the tRNA at the A or P site but varied considerably with the length and structure of the probe side chain. When the distance from the pyrimidine C-5 atom to the azide group was 23 A, 42-45% cross-linking was obtained at each site, but when the distance was decreased to 18 A, only 7-12% was found. Placing an S-S bond in the center of the 23-A leash decreased the A-site yield to about half, while insertion of a CONH group decreased A-site cross-linking about 8-fold. P-site cross-linking was more sensitive to mercaptan quenching (50% at 0.5 mM) than was that at the A site (50% at greater than 2.0 mM) but both were partially shielded from solvent.(ABSTRACT TRUNCATED AT 250 WORDS)