title: Random mutagenesis of yeast 25S rRNA identify bases critical for 60S subunit structural integrity and function.
abstract: In yeast Saccharomyces cerevisiae, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During translation initiation, the 60S subunit joins the 40S initiation complex, producing the 80S initiation complex. During elongation, the 60S subunit binds the CCA-ends of aminoacyl- and peptidyl-tRNAs at the A-loop and P-loop, respectively, transferring the peptide onto the α-amino group of the aminoacyl-tRNA. To study the role of 25S rRNA in translation in vivo, we randomly mutated 25S rRNA and isolated and characterized seven point mutations that affected yeast cell growth and polysome profiles. Four of these mutations, G651A, A1435U, A1446G and A1587G, change a base involved in base triples crucial for structural integrity. Three other mutations change bases near the ribosomal surface: C2879U and U2408C alter the A-loop and P-loop, respectively, and G1735A maps near a Eukarya-specific bridge to the 40S subunit. By polysome profiling in mmslΔ mutants defective in nonfunctional 25S rRNA decay, we show that some of these mutations are defective in both the initiation and elongation phases of translation. Of the mutants characterized, C2879U displays the strongest defect in translation initiation. The ribosome transit-time assay directly shows that this mutation is also defective in peptide elongation/termination. Thus, our genetic analysis not only identifies bases critical for structural integrity of the 60S subunit, but also suggests a role for bases near the peptidyl transferase center in translation initiation.
authors.affiliation: Molecular Cellular and Developmental Biology Program; Division of Biology; Kansas State University; Manhattan, KS USA.
authors.forename: Naoki
authors.initials: N
authors.lastname: Nemoto
authors.affiliation: Molecular Cellular and Developmental Biology Program; Division of Biology; Kansas State University; Manhattan, KS USA.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Udagawa
authors.affiliation: Molecular Cellular and Developmental Biology Program; Division of Biology; Kansas State University; Manhattan, KS USA.
authors.forename: Wasimul
authors.initials: W
authors.lastname: Chowdhury
authors.affiliation: Insititute for Virus Research; Kyoto University; Kyoto, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kitabatake
authors.affiliation: Laboratory of Gene Regulation and Development; Eunice Kennedy Shriver NICHD; National Institutes of Health; Bethesda, MD USA.
authors.forename: Byung-Shik
authors.initials: BS
authors.lastname: Shin
authors.affiliation: Molecular Cellular and Developmental Biology Program; Division of Biology; Kansas State University; Manhattan, KS USA.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hiraishi
authors.affiliation: Molecular Cellular and Developmental Biology Program; Division of Biology; Kansas State University; Manhattan, KS USA; Arthropod Genomics Center; Division of Biology; Kansas State University; Manhattan, KS USA.
authors.forename: Suzhi
authors.initials: S
authors.lastname: Wang
authors.affiliation: Molecular Cellular and Developmental Biology Program; Division of Biology; Kansas State University; Manhattan, KS USA.
authors.forename: Chingakham Ranjit
authors.initials: CR
authors.lastname: Singh
authors.affiliation: Molecular Cellular and Developmental Biology Program; Division of Biology; Kansas State University; Manhattan, KS USA; Arthropod Genomics Center; Division of Biology; Kansas State University; Manhattan, KS USA.
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Brown
authors.affiliation: Insititute for Virus Research; Kyoto University; Kyoto, Japan.
authors.forename: Mutsuhito
authors.initials: M
authors.lastname: Ohno
authors.affiliation: Molecular Cellular and Developmental Biology Program; Division of Biology; Kansas State University; Manhattan, KS USA.
authors.forename: Katsura
authors.initials: K
authors.lastname: Asano
keywords: 25S rRNA; 60S subunit; ribosome; subunit joining; translation initiation; yeast
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