Ribosome biogenesis and function are ubiquitous processes central to cell growth and regulation. The long-term goal of this work is to understand the mechanisms of coordinate expression and assembly of yeast, ribosomal protein and ribosomal RNA and the mechanisms whereby ribosomes catalyze protein synthesis. The mechanisms of equimolar synthesis of ribosomal proteins and rRNAs or of coordinate changes in their synthesis are not yet fully understood. Ribosomal RNAs, transcribed in the nucleolus, and ribosomal proteins, synthesized in the cytoplasm, assemble into ribosomes within the nucleolus. Little is known about the structure and function of nonribosomal nucleolar molecules or about the positions or function of individual ribosomal molecules in the assembly pathway or within the mature ribosome. Cloned genes for three ribosomal proteins, rp59, L16, and L1, and nucleolar protein genes will be utilized to investigate the following specific aims: (1) Expression of CRY2 encoding rp59 is feedback inhibited by rp59, suggesting a mechanism for coupling ribosome synthesis and assembly. Steps at which CM expression is repressed are being determined. Cis-acting repressor sites and transacting repressors are being defined. (2a) By immunoelectron microscopy L16 was localized near the central protuberance of the 60S subunit. Biochemical and genetic experiments are planned to map L16 at higher resolution and to define nearest neighbors of L16. (b)The effect of rpll6 conditional lethal mutations on ribosome assembly and function will be assayed to assess the role of L16 in these processes. (3)Ribosomal protein L1 (or its homologues) and 5S rRNA form an RNP complex that is a precursor to ribosome assembly in mammals, Xenopus, and bacteria. Using wild type d mutant RPL1 and 5S rRNA genes and antiserum specific for the RNP, we will define the pathway of 5S RNP biogenesis. Domains of Ll and 5S rRNA necessary for their binding to each other will be mapped. Models will be tested whereby 5S rRNA synthesis may be coupled to ribosomal protein synthesis. (4) Nucleolar protein genes will be cloned and mutant alleles isolated to assess functions of nucleolar proteins.
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