The mechanisms of the regulation of biosynthesis of ribosomes in the yeast Saccharomyces cerevisiae will be investigated, utilizing recombinant DNA molecules that contain ribosomal protein genes whose expression can be readily assayed in vivo. Our studies will focus on the transcriptional and translational regulatory mechanisms governing expression of the RP59 (CRY) genes, encoding sensitivity or resistance to the antibiotic cryptopleurine, and the RP39 genes. RP39 lacZ gene fusions will be used to conveniently assay RP39 expression. The post transcriptional mechanisms for maintaining the balanced production of ribosomal proteins will be studied by assaying the subcellular location, physical state, and translation of excess rp mRNA synthesized upon galactose induction of GAL1 RP39 or GAL1 RP59 promoter fusions. Mutants overproducing rp39 or rp59 proteins will be isolated to genetically define this regulatory mechanism. Cell free protein synthesizing extracts of yeast will be used to biochemically identify translational regulatory molecules. The relationship between ribosome assembly and control of ribosomal protein gene expression will be investigated by assaying rp synthesis and ribosome assembly subsequent to inhibition of expression of the RP39 or RP59 genes. Site directed and random mutations will be generated in vitro in DNA upstream of the CRY-S1 (RP59A) and RP39A-1acZ genes to further delineate the nature of ribosomal protein gene promoters. DNase protection and filter binding experiments will be performed with these DNAs to ascertain which if any sequences within the promoters bind to proteins. These proteins will then be identified by further biochemical and genetic approaches. The expression of each of the duplicated RP39 and RP59 genes will be assayed using allele specific hybridization probes to determine whether the genes are differentially expressed under any growth conditions. The expression of the RP39 and RP59 genes during shifts in cellular growth rates will be assayed to determine the mechanism of coordinate rp gene expression under these conditions. These studies should further our understanding of coordinate gene expression, as well as the means by which cellular growth rate is coordinated with the synthesis and assembly of the translational apparatus.
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