We will continue to explore the mechanisms involved in transcription termination by RNA polymerase l in the yeast, Saccharomyces cerevisiae. At present we know the minimal DNA sequences needed for efficient termination in vitro, we know that a protein, Reblp, binds to this sequence and acts as a terminator protein, and we have evidence that an RNA 3' processing activity associates with termination. We have proposed a model for termination that incorporates all of these elements and we will test this model in as many ways as possible. For future studies we have developed an in vivo color assay in which yeast colonies change from white to red when polI termination is active. Using this assay we will more rigorously define both DNA sequences and proteins required for termination in the living cell. This will involve doing genetic screens for proteins involved in termination as well as further defining the regions of known proteins (such as polI itself and Reblp) which are essential for termination. The RNA 3' end processing activity associated with poll is similar in action but physically distinct from the polII elongation factor, SII (TFIIS). We will purify the polI associated processing factor, clone its gene, and determine its role, if any, in the termination process. An essential part of the poll termination model is the proposition that Reblp bound to its DNA site acts as a pausing signal that is specific for poll. To determine how this occurs we intend to determine the crystal structure of the Reblp DNA binding domain co-crystallized with its cognate DNA site. Differential termination and/or blocks to elongation are used as control mechanisms for numerous human oncogenes as well as for HIV-1. At present poll termination is probably the best understood and most accessible termination system for eukaryotic RNA polymerases. We believe that insights gained in this model system will likely aid in understanding termination by other eukaryotic RNA polymerases as well.
