Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1946908 | Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms | 2009 | 8 Pages |
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.
Keywords
MBPShine–DalgarnoIPTGDTTLBEX-Gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideS/DONPGBSAbovine serum albuminBlastisopropyl β-D-thiogalactopyranosideRNA–protein interactionsuppressor mutationDissociation constantdithiothreitolNon-canonicalLuria–Bertaniribosomal proteinmaltose-binding proteinoptical density
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Authors
James J. Schweppe, Chaitanya Jain, Susan A. White,