Role of ribosomal protein S12 in discrimination of aminoacyl-tRNA.

Ribosomes of streptomycin-resistant strains (&r-R) translate genetic messages with fewer errors than ribosomes of streptomycin-sensitive (S&S) strains. I have examined whether the increased level of tRNA discrimination by &r-R ribosomes, relative to Str-S ribosomes, occurs in an initial discrimination step before GTP hydrolysis during enzymatic tRNA binding, or in a second discrimination, or proofreading, step following GTP hydrolysis. Str-S or &r-R ribosomes, programmed with polyuridylate (poly(U)) and carrying 7%acetylphenylalanyl-tRNA in the P-site, were allowed to react with elongation factor Tu, GTP, and LeutRNAzL”” (codon CW) or Ile-tRNA (codon AW). In the presence of poly(U), these noncognate tRNAs stimulated the hydrolysis of GTP at a much greater rate than the rate of amino acid incorporation into dipeptides, confirming a previous report that rejection of noncognate tRNAs by ribosomes occurs following GTP hydrolysis. tRNA discrimination by &r-R ribosomes is greater than that of Str-S ribosomes in the first discrimination step, as evidenced by lower rates of GTP hydrolysis, and in the second discrimination step, as evidenced by higher GTP/dipeptide ratios for noncognate tRNAs. The addition of streptomycin essentially abolished the second discrimination step, resulting in GTP/ dipeptide ratios of approximately 1 for Str-S ribosomes. In addition, streptomycin appeared to increase the rate of GTP hydrolysis for noncognate tRNAs. Because these results parallel the known effects of Str-R mutations and of streptomycin on the fidelity of protein synthesis in vivo, tRNA discrimination probably takes place in two steps in vivo, one before and one after GTP hydrolysis. Ribosomal protein S12 influences tRNA selection at both discrimination steps.