Distinct functional classes of ram mutations in 16S rRNA

Effects of 16S A-site mutations on EF-Tu-dependent GTP hydrolysis and RF2-dependent termination. (A,B) 70S initiation complexes (70SIC) programmed with either cognate UUU or near-cognate CUU in the A site were rapidly mixed with EF-Tu•[γ³²P]GTP•Phe-tRNA^Phe, and rates of GTP hydrolysis were determined. Shown are apparent rates for cognate (A) and near-cognate (B) reactions plotted vs. 70SIC concentration. Data were fit to the equation k_app = k_cat[70SIC]/(K_M+[70SIC]), yielding the kinetic parameters shown in Table 1. (C) Ribosomes (0.2 µM) containing Ac[³⁵S]Met-tRNA^Met in the P site and codon UAA in the A site were rapidly mixed with RF2, and rates of Ac[³⁵S]Met-tRNA^Met hydrolysis were determined. Shown are apparent rates plotted as a function of RF2 concentration. Data were fit to the quadratic equation k_app = k_cat{(A + B + K_M) – [(A + B + K_M)² – 4AB]^1/2}/(2A), where A and B represent the total concentrations of ribosomal complex and RF2, respectively, in the reaction. This yielded the k_cat and K_M parameters shown in Table 2. Control, ○ and solid lines; C1054U, □ and long-dashed lines; C1054A, ⋄ and medium-dashed lines; C1200U, △ and short-dashed lines; G1491A, ▽ and dotted lines.