title: Compensatory mutations in the L30e kink-turn RNA-protein complex.
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.
authors.affiliation: Department of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Schweppe
authors.forename: Chaitanya
authors.initials: C
authors.lastname: Jain
authors.forename: Susan A
authors.initials: SA
authors.lastname: White
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D005800:Genes, Fungal; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D046911:Macromolecular Substances; D012331:RNA, Fungal; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C035201:ribosomal protein L30