Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus

Association of a/eIF2 with different binding partners. (A) The k_on values for a/eIF2₅₆₅ binding to Met-tRNAi_F^Met (■) and to 5′-P₃-2508sh_F RNA (▼) were determined by titration of the respective k_app values obtained at different concentrations of a/eIF2₅₆₅. (B) The k_on values for Met-tRNAi_F^Met (■) and 5′-P₃-2508sh_F RNA (▼) binding to a 30S•aIF1•aIF1A•a/eIF2₅₆₅-GTP complex were determined by titration of the respective k_app values obtained at different concentrations of the 30S•aIF1•aIF1A•a/eIF2₅₆₅-GTP complex. The charts in A and B show the k_on, k_off, and K_d values determined as described in Materials and Methods. (C) 30S•a/eIF2 binding to Met-tRNAi_F^Met (blue trace) and to equimolar concentrations (see Materials and Methods) of Met-tRNAi_F^Met and 5′-P₃-2508sh RNA (orange trace). Control measurements were performed with unlabeled AUG-mRNA and labeled a/eIF2₅₆₅ (black trace, acceptor only) or with labeled Met-tRNAi_F^Met and unlabeled a/eIF2 (gray trace, donor only). As donor and acceptor were stable under the experimental conditions, the downward slope of the two control curves may reflect photobleaching.