A second-site mutation at glutamate-257 that restores the function of the mutant yeast ribosomal protein L5 containing lysine-270,271→arginine

Lee Chuan C. Yeh, John C. Lee

Research output: Contribution to journalArticle

Abstract

A genetic approach was used to identify interacting regions of yeast ribosomal protein L5 (also known as L1, L1a, or YL3). Previous studies from our laboratory showed that residues K270 and K271 in protein L5 are essential for its function. The mutant L5 protein in which both residues were replaced by arginine residues (K270,271R) exhibited about 80% RNA binding capability compared to the wild-type and the mutant protein was assembled into the 60S ribosomal subunits in vivo. The yeast strain expressing this mutant protein in a homozygous form was lethal (Biochim. Biophys. Acta 1308 (1996) 133-141). In the present study, this non-functional mutant was used to select intragenic suppressors. A spontaneous, intragenic suppressor which contained an E257K substitution (in addition to the primary mutations) was identified. The suppressor protein bound about 60% of yeast 5S rRNA in vitro compared to the wild-type. To gain more insight into the nature of the intragenic suppressor, additional mutant proteins in which E257 was substituted by a variety of amino acids were produced by site-directed mutagenesis. The ability of each mutant protein to bind yeast 5S rRNA in vitro and to suppress the lethal effect of the double K270,271 mutation in vivo were examined. Results suggest communication between two non-contiguous domains on protein L5 and that several factors, such as electrostatic interaction and hydrogen bonding are likely to play a role in this global communication. Mutation studies on E257 alone also reveal that substitutions of this residue in L5 protein could affect cell growth under specified conditions, but a variety of changes could be tolerated without serious deleterious effects. We propose a working model in which E257 is located in a loop and the dynamic as well as the flexibility of this loop is important for L5 function. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)223-232
Number of pages10
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Volume1489
Issue number2-3
DOIs
StatePublished - Dec 23 1999

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Keywords

  • 5S rRNA
  • Intragenic suppressor
  • RNA-protein interaction
  • Ribosomal protein
  • Second-site revertant
  • Yeast

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

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