EMBO Conference on Protein Synthesis and Translational Control

ADESH SAINI
5
Speaker Abstracts
Structural elements in eIF1A regulate AUG selection by controlling distinct
modes of initiator binding to the preinition complex
Jagpreet Nanda 1, Jon Lorsch 1, Alan Hinnebusch 2, Adesh Saini 2
1 Johns Hopkins School of Medicine, United States of America
2 National Institute of Health, United States of America
eIF1A is the eukaryotic ortholog of bacterial initiation factor 1(IF1), but contains an additional
helical domain and long unstructured N- and C-terminal tails (NTT and CTT). We identified a
repeated motif in the CTT (SE-1 and SE-2) that promotes recruitment of the
eIF2·GTP·Met-tRNAiMet ternary complex (TC), and also suppresses initiation at non-AUG
codons. Compound mutations affecting both SEs produce stronger defects in TC recruitment
in vitro and in vivo (Gcd- phenotype), greater UUG initiation in vivo (Sui- phenotype), and
stronger growth defects compared to mutating SEs individually, and the complete elimination
of both SEs is lethal. Remarkably, the elevated UUG initiation and growth phenotypes of SE
mutations are suppressed by overexpressing eIF1, a putative scanning enhancer, and by
mutations in three other segments of eIF1A: the NTT (SI-1) and the structured N- and C-
strands that pack against α2 in the helical domain (SI-2N and SI-2C). Strikingly, SI mutations
also rescue the TC binding/Gcd- defects conferred by SE mutations and a Sui-/Gcd- mutation
in eIF2β. These results indicate that SE and SI elements regulate start codon selection through
opposing effects on TC binding. We envision that TC binds to the scanning-conducive
conformation of the preinitiation complex in a manner that prevents base-pairing between
initiator and P-site triplets (P[out] state), and that the transition to a scanning-arrested
conformation is promoted by perfect codon-anticodon pairing at AUG (P[in] state). SE
elements appear to stabilize TC binding in the P[out] state and thereby promote scanning and
suppress non-AUG initiation, whereas SI elements antagonize TC binding in the P[out] state to
enhance P[in] binding and start codon recognition. Biochemical analysis indicates that SE
elements also stimulate eIF1 dissociation specifically at AUG codons, and loss of this function
likely further enhances non-AUG initiation in SE mutants.