Characterization of Ribosomal Protein L2 and Analysis of Missense Codon Discrimination in the Yeast Saccharomyces cerevisiae

Abstract

Translational fidelity is of vital importance to the ribosome as it strikes an optimal balance between speed and accuracy. The study of factors contributing to the structure and function of the ribosome by assaying effects on fidelity yields important insights into the mechanism of the ribosome. Ribosomal protein L2 is vital to the structure and function of the ribosome with importance in RNA interaction and positioning, ribosomal subunit binding, and peptidyl transfer. Sixteen unique alleles of the RPL2A gene were studied for their effects on translational fidelity and general ribosome function. Mutations at V48D and L125Q of the highly structured globular domain proved generally deleterious to growth, but resistant to anisomycin, and markedly increased -1 programmed ribosomal frameshifting. H215Y, a mutation at the site of interaction with H93 of the peptidyl transferase center (PTC), was both sparsomycin and anisomycin resistant, indicating a functional impact on the PTC structure. Mutations at F185L and W195C border conserved rRNA binding regions and proved hyperaccurate in misincorporation studies. Understanding of fidelity and proper tRNA selection in translation requires analysis of how the ribosome discriminates between cognate, near- cognate, and non-cognate tRNAs. Measuring misreading of several codon substitutions in the context of paromomycin as well as mutations and deletions of contributing elongation factors and ribosome structural elements allowed further understanding of interactions governing tRNA selection and tRNA misincorporation events. A mutant of eEF1Balpha, the nucleotide exchange factor for eEF1A, promoted a general increase in fidelity. Interestingly, a mutated ribosomal protein L5, though distant from the decoding center, also stimulated hyperfidelity in both near- and non-cognate cases. Distinction between near- and non-cognate tRNA interactions on the ribosome was observed in tests using paromomycin, and in studying mutations of eEF1Bgamma with both demonstrating increased misreading of near-cognate codons but hyperaccurate decoding of non-cognate codons.