Abstract #60

Molecular functions of translation initiation factors eIF1 and eIF1A in preinitation complex assembly, ribosomal scanning and AUG recognition. Alan Hinnebusch¹, Christie Fekete¹, Drew Applefield², Yuen Nei Cheung¹, David Maag², Mikkel Algire², Stephen Blakely¹, Nikolay Shirokikh³, Tatyana Pestova³, Jon Lorsch². 1) Laboratory of Gene Regulation & Development, NICHD, NIH, Bethesda, MD; 2) Dept. of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD; 3) Dept. of Microbiology and Immunology, SUNY-Brooklyn, NY.
   Translation initiation begins with recruitment of Met-tRNAi in a ternary complex (TC) with eIF2 and GTP to the 40S ribosomal subunit, to form the 43S preinitiation complex (PIC). The 43S PIC binds mRNA and scans the leader, and AUG recognition triggers GTP hydrolysis and Pi release from eIF2-GDP. In vitro studies implicate eIF1 and eIF1A in these reactions, but their molecular functions are poorly understood. Mutations that impair TC recruitment constitutively derepress GCN4 translation (Gcd- phenotype) in a manner suppressed by overexpressing eIF2 subunits and tRNAi (ie. TC). We identified Gcd- mutations in the C-terminal domain of eIF1A that impair binding of TC and several other initiation factors (eIF3, eIF5 and eIF1), but not eIF1A itself, to native 43S PICs in vivo. Analysis of PIC assembly in vitro with purified components confirms a reduced rate of TC loading with no reduction in eIF1A affinity for 40S subunits, thus implicating the eIF1A CTD in 43S PIC assembly. Mutations in the eIF1A CTD and adjoining helical domain also impair scanning in vitro, as judged by “toe-print” mapping of 43S binding sites on mRNA. This leads to increased selection of UUG start codons, suppressing an AUG start mutation at his4-303 (Sui- phenotype). In vitro, the mutations disrupt an interaction between eIF1A and eIF5 that regulates the transition between an “open” scanning-competent PIC and a “closed” complex containing tightly bound eIF1A arrested at the start codon. Finally, analysis of a Sui- mutation in eIF1 supports the model that GTP hydrolysis, Pi release and scanning-arrest at AUG all require dissociation of eIF1 from the 40S subunit, supporting a critical “gate-keeper” function for eIF1 in AUG recognition.

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