title: Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons.
abstract: The eukaryotic 43S preinitiation complex (PIC) bearing Met-tRNAiMetin a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site ("POUT") to a closed, arrested conformation with TC tightly bound in the "PIN" state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high. Two such substitutions-R116D and R117D-also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed PINstate with a UUG-anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC-mRNA contacts at the entry channel, augmenting the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Jinsheng
authors.initials: J
authors.lastname: Dong
authors.affiliation: Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Colin Echeverría
authors.initials: CE
authors.lastname: Aitken
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Anil
authors.initials: A
authors.lastname: Thakur
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Byung-Sik
authors.initials: BS
authors.lastname: Shin
authors.affiliation: Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 ahinnebusch@nih.gov jon.lorsch@nih.gov.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Lorsch
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; ahinnebusch@nih.gov jon.lorsch@nih.gov.
authors.forename: Alan G
authors.identifier: 0000-0002-1627-8395
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D018387:Codon, Initiator; D040061:Eukaryotic Initiation Factor-5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018387:Codon, Initiator; D040061:Eukaryotic Initiation Factor-5; D046912:Multiprotein Complexes; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C044152:ribosomal protein S3
keywords: initiation; ribosome; translation; uS3; yeast