Protein synthesis in rabbit reticulocytes: Requirements for Met-tRNA_f·40S preinitiation complex formation with AUG-codon and physiological mRNAs

Under standard conditions, in the presence of GTP, highly purified eIF-2 and Co-eIF-2 factor preparations efficiently stimulated AUG-codon dependent but not physiological mRNA-dependent Met-tRNA_f binding to 40S ribosomes. Replacement of GTP by a nonhydrolyzable GTP analog, GMP-PNP, in the above system, gave significant stimulation of Met-tRNA_f binding to 40S ribosomes dependent on physiological mRNAs. Lower but significant stimulation of Met-tRNA_f binding to 40S ribosomes was also observed when GTP was used in the presence of nucleoside 5′-diphosphate kinase (NDK) and ATP. ATP alone in the absence of NDK had no significant effect. This is the first report on the formation of a stable Met-tRNA_f·40S initiation complex dependent on physiological mRNAs and the factor requirements for such complex formation.