Eukaryotic initiator tRNA (tRNAi) contains several highly conserved unique sequence features, but their importance in accurate start codon selection was unknown. Here we show that conserved bases throughout tRNAi, from the anticodon stem to acceptor stem, play key roles in ensuring the fidelity of start codon recognition in yeast cells. Substituting the conserved G31:C39 base pair in the anticodon stem with different pairs reduces accuracy (the Sui- [suppressor of initiation codon] phenotype), whereas eliminating base pairing increases accuracy (the Ssu- [suppressor of Sui-] phenotype). The latter defect is fully suppressed by a Sui- substitution of T-loop residue A54. These genetic data are paralleled by opposing effects of Sui- and Ssu- substitutions on the stability of methionylated tRNAi (Met-tRNAi) binding (in the ternary complex [TC] with eIF2-GTP) to reconstituted preinitiation complexes (PICs). Disrupting the C3:G70 base pair in the acceptor stem produces a Sui- phenotype and also reduces the rate of TC binding to 40S subunits in vitro and in vivo. Both defects are suppressed by an Ssu- substitution in eIF1A that stabilizes the open/POUT conformation of the PIC that exists prior to start codon recognition. Our data indicate that these signature sequences of tRNAi regulate accuracy by distinct mechanisms, promoting the open/POUT conformation of the PIC (for C3:G70) or destabilizing the closed/PIN state (for G31:C39 and A54) that is critical for start codon recognition.
ASJC Scopus subject areas
- Developmental Biology