The normal growth and development of all organisms requires the proper response to a myriad of extracellular stimuli including nutrients, growth factors and hormones. In addition, the cell must also contend with conditions detrimental to viability (e.g., excess heat, toxins). Regardless of the stimuli, one important task of the molecules that transduce these signals is to influence the activity of factors that regulate transcription. Unregulated stimulation of signal transduction cascades or transcription factors underlie several human maladies including cancer. Therefore, elucidating the molecular mechanisms that link signaling pathways to the transcription machinery is key to understanding many disease states. Recent findings indicate that the yeast cyclin-cyclin dependent kinase (CDK) complex Ume3p- Ume5p provides an important connection between the regulatory systems responding to environmental stimuli and the transcription apparatus. First, Ume3p-Ume5p co-localize with the RNA polymerase II holoenzyme, a complex required for transcription initiation suggesting this cyclin-CDK complex directly modifies a component(s) of the basal transcription machinery. Moreover, Ume3p-Ume5p are required for the repression of several genes necessary for the stress response or meiotic development. To relieve this repression, Ume3p is rapidly destroyed in cells exposed to stress (e.g., heat shock) or following entry into the meiotic program. This destruction is important as the failure to destroy Ume3p suppresses meiotic gene expression and alters meiosis I progression. Remarkably, Ume3p destruction is not dependent on the ubiquitin-dependent proteolytic pathway or the 26S proteasome. These findings suggest that Ume3p is controlled through mechanisms different than those that regulate mitotic cyclins. Using a novel selection strategy, three mutants (rut1- 3) have been recovered that are defective in Ume3p destruction in response to heat shock. The experiments described in this proposal will combine genetic, molecular and biochemical approaches to understand both the individual roles of these genes in regulating Ume3p and where they fit into the regulatory system governing meiosis or the stress response.
