Reduced Formation of Initiation Complexes between Met‐tRNA f and 40‐S Ribosomal Subunits in Rabbit Reticulocyte Lysates Incubated at Elevated Temperatures

Rabbit reticulocyte lysates preincubated at 42–45 °C and subsequently assayed for protein synthesis at 37 °C show (a) a decrease in their rate of polypeptide chain initiation and (b) decreased Met‐tRNA f binding to native 40‐S subunits and 80‐S ribosomes. Similarly, the amount of [ 35 S]Met‐tRNA f . 40‐S‐subunit initiation complexes formed in vitro with native 40‐S subunits isolated from supraoptimally heated lysates is only 10–30% of that formed with native 40‐S subunits prepared from control lysates preincubated at 0 °C or 37 °C. This decrease is not due to reduced formation of Met‐tRNA f ternary complex or to increased destruction of this complex, since we find that 0.5 M KCl ribosomal washes extracted from lysates preincubated at 0 °C, 37 °C, or 43–45 °C have almost identical activities in Met‐tRNA f ·GTP· eIF‐2 ternary complex formation and Met‐tRNA f hydrolysis, whether assayed at 37 °C or 45 °C. Similarly, the results of binding assays of [ 35 S]Met‐tRNA f to 40‐S subunits performed at both temperatures show that the control and heated washes, added at either limiting or optimal amounts, stimulate almost equally the binding of Met‐tRNA f to (a) salt‐washed derived 40‐S subunits and (b) native 40‐S subunits, prepared from control or supraoptimally heated lysates. Moreover the factor‐directed binding of Met‐tRNA f by the derived 40‐S subunits is not affected by the presence of either the control or heated post‐ribosomal supernatant. We conclude that at elevated temperatures there is no irreversible inactivation of the Met‐tRNA f binding factor for ternary complex formation and Met‐tRNA f binding to 40‐S subunits in vitro and discuss some possible explanations for the lowered Met‐tRNA f binding observed in vivo .