Conformational changes in the P site and mRNA entry channel evoked by AUG recognition in yeast translation preinitiation complexes |
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| Authors: | Fan Zhang Adesh K Saini Byung-Sik Shin Jagpreet Nanda Alan G Hinnebusch |
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| Institution: | 1.Laboratory of Gene Regulation and Development, NICHD, NIH, Bethesda, MD 20892, USA;2.Department of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh-173212, India;3.Laboratory on the Mechanism and Regulation of Protein Synthesis, NICHD, NIH, Bethesda, MD 20892, USA |
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| Abstract: | The translation preinitiation complex (PIC) is thought to assume an open conformation when scanning the mRNA leader, with AUG recognition evoking a closed conformation and more stable P site interaction of Met-tRNAi; however, physical evidence is lacking that AUG recognition constrains interaction of mRNA with the 40S binding cleft. We compared patterns of hydroxyl radical cleavage of rRNA by Fe(II)-BABE tethered to unique sites in eIF1A in yeast PICs reconstituted with mRNA harboring an AUG or near-cognate (AUC) start codon. rRNA residues in the P site display reduced cleavage in AUG versus AUC PICs; and enhanced cleavage in the AUC complexes was diminished by mutations of scanning enhancer elements of eIF1A that increase near-cognate recognition in vivo. This suggests that accessibility of these rRNA residues is reduced by accommodation of Met-tRNAi in the P site (PIN state) and by their interactions with the anticodon stem of Met-tRNAi. Our cleavage data also provide evidence that AUG recognition evokes dissociation of eIF1 from its 40S binding site, ejection of the eIF1A-CTT from the P-site and rearrangement to a closed conformation of the entry channel with reduced mobility of mRNA. |
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