We have recently discovered that a fragment of the proteasome 19S regulatory particle plays one or more direct roles in RNA polymerase II transcription that is distinct from proteolysis. This exciting finding opens up a new area of research in transcription enzymology. Preliminary results indicate that the fragment of the 19S, which we call the APIS complex, is important for efficient polymerase II elongation and probably also negatively regulates activators such as Gal4. This project is directed towards obtaining a better understanding of several aspects of the role of the APIS complex in poI II transcription.To date, all of the experiments that we have done have focused on the well-characterized GAL system. DNA microarray experiments in yeast are proposed that will evaluate the importance of APIS for expression on a genome-wide scale. Biochemical experiments will be done to characterize the composition of the APIS complex, which is currently poorly defined. Chromatin immunoprecipitation assays in a variety of genetic backgrounds will be employed to determine whether the APIS complex may be involved in the formation of pre-initiation complexes, in addition to stimulating elongation. Similar approaches will be employed to determine if the APIS complex requires direct interactions with an activator in order to be loaded into the transcription complex. In another set of studies, the mechanistic basis of the inhibitory effect of the APIS complex on Gal4 will be probed using a combination of in vivo and in vitro experiments. Finally, we will probe the possible connection between activator mono-ubiquitination and the role of the APIS complex in transcription. This post-translational modification has been very recently discovered to play a critical role in mediating high-level gene expression in at least one case, but the biochemical underpinnings of this phenomenon are unclear. We will test models that activator ubiquitination either stimulates recruitment of the APIS complex to promoters, or that it alters APIS's catalytic activity and protects activators from the inhibitory activities of this complex. It is anticipated that these experiments will shed important new light on this new and exciting aspect of transcription enzymology.
