Genetics and Biochemistry of Ribosome Synthesis

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DESCRIPTION (provided by applicant): The ribosome, the central machinery essential for protein synthesis, obviously plays a key role in cell growth and its synthesis is intimately connected to the regulation of cell proliferation. Indeed, recent studies suggest important roles for regulation of ribosome synthesis in the development and growth of tumor cells. Our goal is to understand how cells regulate the production of ribosomes in response to environmental conditions. Since transcription of ribosomal RNA (rRNA) is central in determining overall synthesis of ribosomes, our work focuses on regulation of rRNA synthesis by RNA Polymerase I (Pol I). We use the yeast Saccharomyces cerevisiae as a model system because of the ease of genetic manipulation and the available knowledge of rRNA synthesis by RNA polymerase I. Previous studies showed that yeast cells change rRNA synthesis rate by two mechanisms. One is by altering the number of active (open) genes, and another is by altering the activity of individual active genes. In the past, we concentrated on studies of mechanisms of initiation of transcription at individual active genes. Although we will continue this line of work, more emphasis will now be given to studies of post-initiation steps and its regulation at individual active genes. It is now known that rRNA modification, processing and some of assembly reactions take place co-transcriptionally. Therefore, it is likely that transcription and processing/assembly of rRNA are coupled and co-regulated. We have recently identified that mutational defects in the Spt4/5 complex, a known elongation factor for mRNA transcription, cause defects in elongation of Pol I. We plan to identify regions within the rRNA gene that require this factor for their transcription and study roles played by this factor using genetic and biochemical approaches. We will isolate mutants with specific defects in Pol I transcription elongation steps. We are especially interested in those mutants with defects in coupling of transcription with rRNA processing/assembly. In relation to this goal, we have unpublished observations showing an apparent dependence of 18S rRNA synthesis (and/or 40S subunit assembly) on 5.8S-25S rRNA synthesis (and/or 60S subunit assembly). We plan to determine whether 18S rRNA transcription itself, rather than 40S subunit assembly, is dependent on 5.8S-25S rRNA transcription (and/or 40S subunit assembly), and then to study the mechanism involved. Finally, we plan to study the mechanism that controls switching between active and inactive states of rRNA genes. We will compare RNA and protein components associated with active genes with those associated with inactive genes in order to build an understanding of the mechanism involved in switching. We also plan to isolate mutants defective in switching and study these mutants to identify the mechanism. We expect that the proposed work will make significant contributions to our understanding of transcription of rRNA genes and its regulation.