Selection of the AUG start codon is a key step in translation initiation requiring hydrolysis of GTP in the eIF2•GTP•Met-tRNA iMet ternary complex (TC) and subsequent P i release from eIF2•GDP•P i . It is thought that eIF1 prevents recognition of non-AUGs by promoting scanning and blocking P i release at non-AUG codons. We show that Sui − mutations in Saccharomyces cerevisiae eIF1, which increase initiation at UUG codons, reduce interaction of eIF1 with 40S subunits in vitro and in vivo, and both defects are diminished in cells by overexpressing the mutant proteins. Remarkably, Sui − mutation ISQLG 93-97 ASQAA (abbreviated 93-97) accelerates eIF1 dissociation and P i release from reconstituted preinitiation complexes (PICs), whereas a hyperaccuracy mutation in eIF1A (that suppresses Sui − mutations) decreases the eIF1 off-rate. These findings demonstrate that eIF1 dissociation is a critical step in start codon selection, which is modulated by eIF1A. We also describe Gcd − mutations in eIF1 that impair TC loading on 40S subunits or destabilize the multifactor complex containing eIF1, eIF3, eIF5, and TC, showing that eIF1 promotes PIC assembly in vivo beyond its important functions in AUG selection.[Keywords: AUG selection; Saccharomyces cerevisiae; translation initiation; eIF1] Supplemental material is available at http://www.genesdev.org.

Start codon recognition is a crucial event in the initiation of protein synthesis. To gain insight into the mechanism of start codon recognition in eukaryotes, we used a yeast reconstituted initiation system to isolate the step of Met-tRNA i d eIF2 d GTP ternary complex (TC) binding to the 40S subunit. We examined the kinetics and thermodynamics of this step in the presence of base changes in the mRNA start codon and initiator methionyl tRNA anticodon, in order to investigate the effects of base pairing and sequence on the stability of the resulting 43S d mRNA complex. We observed that the formation of three base pairs, rather than their identities, was the key determinant of stability of TC binding, indicating that nothing is inherently special about the sequence AUG for this step. Surprisingly, the rate constant for TC binding to the 40S subunit was strongly codon dependent, whereas the rate constant for TC dissociation from the 43SdmRNA complex was not. The data suggest a model in which, after the initial diffusion-limited encounter of TC with the 40S subunit, the formation of three matching start codon/anticodon base pairs triggers a conformational change that locks the complex into a stable state. This induced-fit mechanism supports the proposal that initiation codon recognition by the 43S complex induces a conformational change from an open state to a closed one that arrests movement along the mRNA.

Eukaryotic translation initiation factor (eIF) 1 is a central mediator of start codon recognition. Dissociation of eIF1 from the pre-initiation complex allows release of phosphate from the G-protein factor eIF2, triggering downstream events in initiation. Mutations that weaken binding of eIF1 to the pre-initiation complex decrease the fidelity of start codon recognition (Sui− phenotype) by allowing increased eIF1 release at non-AUG codons. Consistent with this, over-expression of these mutant proteins suppresses their Sui− phenotypes. Here, we have examined mutations at the penultimate residue of eIF1, G107, that produce Sui− phenotypes without increasing the rate of eIF1 release. We provide evidence that, in addition to its role in gating phosphate release, dissociation of eIF1 triggers conversion from an open, scanning-competent state of the pre-initiation complex to a stable, closed one. We also show that eIF5 antagonizes binding of eIF1 to the complex and that key interactions of eIF1 with its partners are modulated by the charge at and around G107. Our data indicate that eIF1 plays multiple roles in start codon recognition and suggest that prior to AUG recognition it prevents eIF5 from binding to a key site in the pre-initiation complex required for triggering downstream events.

Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui _ .No small molecules are known that affect the fidelity of start codon recognition. Such compounds would be useful tools for probing the molecular mechanics of translation initiation and its regulation. To find compounds with this effect, we set up a high-throughput screen using a dual luciferase assay in S. cerevisiae. Screening of over 55,000 compounds revealed two structurally related molecules that decrease the fidelity of start codon selection by approximately twofold in the dual luciferase assay. This effect was confirmed using additional in vivo assays that monitor translation from non-AUG start codons. Both compounds increase translation of a natural upstream open reading frame previously shown to initiate translation at a UUG. The compounds were also found to exacerbate increased use of UUG as a start codon (Sui _ phenotype) conferred by haploinsufficiency of wild-type eukaryotic initiation factor (eIF) 1, or by mutation in eIF1. Furthermore, the effects of the compounds are suppressed by overexpressing eIF1, which is known to restore the fidelity of start codon selection in strains harboring Sui _ mutations in various other initiation factors. Together, these data strongly suggest that the compounds affect the translational machinery itself to reduce the accuracy of selecting AUG as the start codon.