Characterization of the Ribosomal Protein L30 RNA-Protein Interactions Using a Two-Plasmid Reporter System

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

†Department of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010

*Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Coral Gables, FL 33124

In the Saccharomyces cerevisiae L30 system, the ribosomal protein L30 negatively autoregulates its own expression in two ways: L30 protein binds its pre-mRNA to inhibit splicing and its mature mRNA to repress translation. The L30 RNA target contains the kink-turn (K-turn) structural motif, a three-nucleotide bulge flanked by canonical and non-cannonical stems. Each K-turn nucleotide was systematically mutated to the three non-wild-type bases and tested for binding activity with L30 protein using a quantitative in vivo reporter assay. Five RNA mutants, G11A, G11U, A55G, G56U, and A57G, in which L30 protein binding was diminished but not abolished, were identified as candidates for suppressor screening. Suppressors are gain of function mutants, that is, mutant L30 proteins that bind mutant RNA. Using a random mutagenenic PCR protocol, an L30 mutant DNA library was generated with more than 125,000 different genotypes. This random L30 library was used to screen for suppressors of the five RNA mutants, and presented here are the results of that screening.