Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNA(Met) and AUG selection.

Genes & Development
Jinsheng DongAlan G Hinnebusch

Abstract

High-resolution structures of bacterial 70S ribosomes have provided atomic details about mRNA and tRNA binding to the decoding center during elongation, but such information is lacking for preinitiation complexes (PICs). We identified residues in yeast 18S rRNA critical in vivo for recruiting methionyl tRNA(i)(Met) to 40S subunits during initiation by isolating mutations that derepress GCN4 mRNA translation. Several such Gcd(-) mutations alter the A928:U1389 base pair in helix 28 (h28) and allow PICs to scan through the start codons of upstream ORFs that normally repress GCN4 translation. The A928U substitution also impairs TC binding to PICs in a reconstituted system in vitro. Mutation of the bulge G926 in h28 and certain other residues corresponding to direct contacts with the P-site codon or tRNA in bacterial 70S complexes confer Gcd(-) phenotypes that (like A928 substitutions) are suppressed by overexpressing tRNA(i)(Met). Hence, the nonconserved 928:1389 base pair in h28, plus conserved 18S rRNA residues corresponding to P-site contacts in bacterial ribosomes, are critical for efficient Met-tRNA(i)(Met) binding and AUG selection in eukaryotes.

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Citations

Jul 23, 2013·Nature·Ivan B Lomakin, Thomas A Steitz
Oct 18, 2011·Nature Structural & Molecular Biology·Byung-Sik ShinThomas E Dever
Jan 2, 2009·The Journal of Biological Chemistry·Jonathan D Dinman
Nov 4, 2009·Molecular and Cellular Biology·Changyi A LinHeather L True
Sep 3, 2011·Microbiology and Molecular Biology Reviews : MMBR·Alan G Hinnebusch
Feb 7, 2014·Annual Review of Biochemistry·Alan G Hinnebusch
Nov 21, 2009·FEBS Letters·Sarah E Kolitz, Jon R Lorsch
May 10, 2017·Nucleic Acids Research·Jorge Pedro López-AlonsoSean R Connell

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