Compensatory mutations in the L30e kink-turn RNA-protein complex

James J. Schweppe, Chaitanya Jain, Susan A. White

Research output: Contribution to journalArticle

Abstract

The S. cerevisiae ribosomal protein L30e is an autoregulatory protein that binds to its own pre-mRNA and mature mRNA to inhibit splicing and translation, respectively. The L30e RNA-binding element is a stem-asymmetric loop-stem that forms a kink-turn. A bacterial genetic system was designed to test the ability of protein variants to repress the expression of reporter mRNAs containing the L30e RNA-binding element. Initial screens revealed that changes in several RNA nucleotides had a measurable effect on repression of the reporter by the wild type protein. RNA mutants that reduce repression were screened against libraries of randomly mutagenized L30e proteins. These screens identified a glycine to serine mutation of L30e, which specifically restores activity to an RNA variant containing a U that replaces a helix-capping G. Similarly, an asparagine to alanine mutation was found to suppress a substitution at a position where the L30e RNA nucleotide extends out into the protein pocket. In addition, a compensatory RNA mutation within a defective RNA variant was found. The identification of these suppressors provides new insights into the architecture of a functional binding element and its recognition by an important RNA-binding protein.

Original languageEnglish (US)
Pages (from-to)469-476
Number of pages8
JournalBiochimica et Biophysica Acta - Gene Regulatory Mechanisms
Volume1789
Issue number6-8
DOIs
StatePublished - Jun 2009

Keywords

  • Kink-turn RNA
  • L7Ae superfamily
  • Ribosomal protein
  • RNA-protein interaction
  • Suppressor mutation
  • Two-plasmid screen

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Genetics
  • Molecular Biology
  • Structural Biology

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