Fig. 2: Role of the G–C base-pairs clusters in Met-tRNAiMet conformation.
a Sequence diagram of the S. cerevisiae Met-tRNAiMet with initiator-specific base pairs colored in orange and modified nucleotides marked with asterisks. b Overview of the conformation adopted by Met-tRNAiMet on the 80S-IC complex with the initiator-specific G–C base pairs colored in orange. c Superposition of Met-tRNAiMet (green) with a P site classical-state elongation tRNA (gray) reveals a distorted configuration of the acceptor stem of Met-tRNAiMet when compared with an elongation peptidyl-tRNA. This distortion prevents the 74CCA76-Met end of Met-tRNAiMet from reaching the peptidyl transferase center (PTC, blue) on the 60S. d In contrast, the ASL of Met-tRNAiMet has reached its final position when compared with an elongation tRNA in the P site. e The arginine residue 955 at DIV of eIF5B specifically recognizes the Hoogsteen edge of the base of nucleotide G70 of Met-tRNAiMet (red arrow, R955). f Cryo-EM density for side chains and individual bases in the area around eIF5B R955. On the right, chemical diagram with contacts and distances indicated. g–i Yeast growth and GCN4-lacZ reporter assays of strains lacking eIF5B or expressing wild-type or R955A mutant. Cells were spotted on minimal (SD) or starvation (3-AT) medium (g) or assayed for ß-galactosidase activity following introduction of a wild-type GCN4-laZ reporter (h) or a derivative in which an extended version of uORF1 overlaps, out-of-frame, the GCN4 start codon (i). Results are the average and standard deviation of six independent transformants combined from two independent experiments with three replicates each; p values were calculated using two-sided Student’s t test. The p values for panel (h) are eIF5B vs. eIF5BΔ: 7.57349 × 10−12, eIF5B vs. eIF5B-R955A: 6.30158 × 10−9 and for panel (i) are eIF5B vs. eIF5BΔ: 1.23215 × 10−5, eIF5B vs. eIF5B-R955A: 1.3313 × 10−6.
