Dominant-Negative Mutants of Ytm1p, an Essential Non-Ribosomal Protein in Saccharomyces cerevisae

Ribosome assembly is a complicated process involving the maturation of rRNA and it’s association with ribosomal proteins. Non-ribosomal proteins are also involved in coordinating the assembly of ribosomes by associating with the pre-rRNPs (ribonuclear protein particles) and then later dissociating. Ytm1p, an essential and conserved gene, was found to be involved in the maturation of the 66S pre-rRNP in both classic genetic studies and state of the art proteomic studies. Ytm1p is one of 17 known proteins involved in ribosome assembly that contains WD40 repeat structures. However, none of these proteins have been studied in great detail. Thus, studying the structure and function of Ytm1p can provide a model for the behavior of the WD40 repeat protein in multi-molecular complexes. Ytm1p, with its seven WD40 repeats, may be used as a scaffolding protein to nucleate and coordinate the assembly of protein-protein complexes or protein-RNA complexes used in the synthesis and maturation of the 66S pre-ribosomes. Specific sites on the protein may be involved in the scaffolding activity of Ytm1p. By mutating specific amino acids in the Ytm1p protein and studying the specific effects of the mutations, one can infer information on the function of the protein. Single point mutations were created in YTM1 using error-prone PCR and the genes were introduced into plasmids via gap repair. Mutant alleles of ytm1 were placed under the control of the inducible GAL1 promoter in order to screen for dominant-negative mutations. Seven mutants were identified in this screen: four strong dominant-negative mutants which inhibited growth of the yeast cells on media containing galactose, and three weaker mutants which only slowed growth on galactose media. These mutants were screened for defects in the synthesis of rRNA and will be screened for defects in polysome profiles and cell cycle progression. Characterizing the specific phenotypes of these dominant-negative mutations and their effects on the synthesis of the 60S ribosomal subunit will help to identify relationships between the structure of the Ytm1p and its function.