期刊
NUCLEIC ACIDS RESEARCH
卷 48, 期 18, 页码 10280-10296出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkaa761
关键词
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资金
- Intramural Research Program of the National Institutes of Health
In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2 alpha with mRNA, including the -3 nucleotide of the 'Kozak' context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui(-) cells (Ssu(-) phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2.GTP.Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2 alpha-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui(-) phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd(-) phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2 alpha with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.
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