Reduced formation of initiation complexes between Met-tRNAf and 40-S ribosomal subunits in rabbit reticulocyte lysates incubated at elevated temperatures. Activity of the Met-tRNAf binding factor.

Abstract

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-tRNAf binding to native 40-S subunits and 80-S ribosomes. Similarly, the amount of [35S]Met-tRNAf. 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-tRNAf 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-tRNAf·GTP· eIF-2 ternary complex formation and Met-tRNAf hydrolysis, whether assayed at 37 °C or 45 °C. Similarly, the results of binding assays of [35S]Met-tRNAf 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-tRNAf 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-tRNAf 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-tRNAf binding factor for ternary complex formation and Met-tRNAf binding to 40-S subunits in vitro and discuss some possible explanations for the lowered Met-tRNAf binding observed in vivo.