The most actively transcribed genes in any organism are those coding for the ~37S pre-ribosomal RNAs. In eukaryotes, this is accomplished by RNA polymerase I and additional factors. How these interact is of fundamental importance. In vivo analysis of the role of the rDNA transcription factor UBF (upstream binding factor) in catalysis of rRNA synthesis in the yeast Schizosaccharomyces pombe is proposed. To facilitate these studies the S. pombe UBF gene is being cloned. The first transcription extract capable of supporting accurate initiation on an S. pombe rDNA mini-gene is now successfully established, and it is sensitive to the action of a vertebrate rDNA enhance which specifically interacts with UBF. Activation of the rDNA promoter in this S-100 extract is also responsive to regulatory elements in the pombe intergenic spacer (from ~-1800 to ~-850 that apparently act by stably binding RNA polymerase I transcriptional factors. Factors interacting with essential rDNA promoter and intergenic regulatory sequences will be analyzed. Our two experimental aims include: 1.) Analysis of the activity of the S. pombe RNA polymerase I transcription factor UBF in vivo and in vitro; 2.) Characterization of the role of regulatory sequences in the S. pombe rDNA spacer in transcriptional activation of the rDNA promoter and identification of interacting factors. %%% Every cell in an organism has millions of ribosomes, the complexes where proteins are synthesized. The function of these complexes is absolutely required for all cells, since proteins are key determinants of cellular structure and functions. The ribosomes re composed of both protein and nucleic acid components. My laboratory is studying how the essential nucleic acid components of these complexes, called the ribosomal RNAs, are produced. Our approach incorporates both genetic and biochemical analysis of requirements for the production of the ribosomal RNAs and analysis of the means by which a cell regulates this production.
