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 language | English (US) |
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Pages (from-to) | 469-476 |
Number of pages | 8 |
Journal | Biochimica et Biophysica Acta - Gene Regulatory Mechanisms |
Volume | 1789 |
Issue number | 6-8 |
DOIs | |
State | Published - 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