Identification by affinity labeling of potential sites in 23-S rRNA interacting with the 3' end of tRNA.

A derivative of tRNAPhe carrying at its 3' end a photolyzable group was chemically synthesized by coupling p-azidobenzoylglycylhydrazide to periodate-oxidized tRNA. The reaction converts the 3'-terminal ribofuranoside residue into a six-membered ring. The binding of p-azidobenzoylglycylhydrazide-tRNA to 70-S ribosomes resembles the binding of Phe-tRNA in its requirement for poly(U) and Mg2+ dependence. Irradiation at wavelengths greater than 300 nm of complexes of p-azidobenzoylglycylhydrazide-tRNA and 70-S ribosomes results in the covalent binding of approximately 20% of the reversibly bound tRNA to the ribosomes. The labeling occurs predominantly at a single site in 23-S rRNA within the sequence included in the 18-S fragment. To identify the modified sequence, an RNase A digest of 23-S rRNA was fractionated on a hydrophobic matrix (Porapak Q) and adsorbed and non-adsorbed fractions were compared by fingerprint analysis. A relatively intense spot was observed in the expected region in the fingerprint of the adsorbed fraction. The presence of an unusual nucleotide in the sequence of this oligonucleotide was demonstrated by a mobility-shift analysis of a partial nuclease P1 digest. The sequence derived is G*-A-A-G-C (the asterisk denoting the site of modification). Sequences at six positions in the 23-S rRNA are compatible with the sequence of the oligonucleotide determined. Of the six positions five are located within nucleotides 1467-1887. This region of approximately 400 nucleotides is likely to include the sequence interacting with the 3' end of tRNA and possibly forming part of the peptidyl-transferase center.