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Dr. Alan G.Hinnebush
Ph.D.
Head, Program in Cellular Regulation and Metabolism
Chief, Laboratory of Gene Regulation and Development
Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health

"Structural elements in eIF1A regulate AUG selection by controlling distinct modes of initiator tRNA binding to the preinitiation complex."

听

Adesh K. Saini鹿, Jagpreet S. Nanda虏, Jon R. Lorsch虏 and Alan G. Hinnebusch鹿

Laboratory of Gene Regulation and Development, NICHD, NIH, Bethesda, MD 20892

听

eIF1A is the eukaryotic ortholog of bacterial initiation factor 1(IF1), but contains a helical domain and long unstructured N- and C-terminal tails (NTT and CTT) not present in IF1. We identified a repeated motif in the CTT (SE1 and SE2) that promotes ribosomal recruitment of initiator tRNAMet in the ternary complex (TC) with eIF2-GTP, and also blocks non-AUG selection by the scanning preinitiation complex. Compound mutations of both SEs produce stronger defects in TC recruitment in vitro and in vivo (Gcd- phenotype), greater selection of UUG versus AUG start codons (Sui- phenotype), and stronger growth defects (Slg- phenotypes) in vivo than does mutating each individually, and elimination of both SEs is lethal. Remarkably, the Sui-听 and Slg-听 phenotypes of SE mutations are suppressed by overexpressing eIF1, a putative scanning enhancer, and also by mutations in three other segments of eIF1A: the NTT (SI1) and the structured N- and C- strands that pack against 伪2 in the helical domain (SI2N and SI2c). Strikingly, mutating each SI element also ameliorates the TC binding defects conferred by SE mutations in vitro and in vivo. In fact, the SI2N and SI2c mutations increase TC loading in vitro above the wild-type level. These results argue strongly that SE and SI elements regulate AUG selection by exerting opposing effects on TC binding. Whereas SE elements appear to stabilize TC binding in a scanning-conducive conformation that is incompatible with start codon recognition, SI2c opposes this mode of TC binding, and SI2N and SI1 stabilize TC binding in a distinct, scanning-incompatible conformation that enables eIF1 release and AUG selection.