This is a supplemental application for our ongoing NIH grant GM068887. The goal of this grant is to characterize the molecular mechanisms involved in the coordination of RNA polymerase II transcription and mRNA 3' end processing. We have identified several new points of interaction between transcription and cleavage/polyadenylation factors which suggest that the presence of processing factors at the promoter might affect the efficiency and/or specificity of transcription initiation. This may serve as a mechanism to insure the proper loading of processing factors onto the transcriptional complex, and in turn, the subsequent ? polyadenylation of the transcript, which is essential for optimal export, translation, and turnover of mRNA. To investigate this issue, we propose the following specific aims: ? 1. What is the role ofSsu72 in early transcription events? Ssu72 is directly involved in 3' end cleavage of mRNA as subunit of Cleavage/Polyadenylation Factor (CPF), but also affects the activity of TFIIB in ? initiation. Our recent data indicates that depletion of Ssu72 causes a severe defect on transcription in vitro, and that Ssu72 is an RNAP II CTD phosphatase with preference for serine-5 of the CTD repeat. We will use assays that discriminate early events in transcription to define the precise role of Ssu72 in transcription and seek additional targets of the phosphatase activity of Ssu72. ? 2. How is the activity ofSsu72 regulated? Ssu72 interacts in vitro with TFIIB, RNAP II, and the Pta1 ? subunit of CPF. Our hypothesis is that these interactions regulate the activity of Ssu72 in transcription or 3' end processing. We will determine whether these interactions occur in vivo, and then investigate how disrupting these interactions affects Ssu72 function. ? 3. How does Swd2 function in RNAP II termination? Depletion of the CPF subunit Swd2 has no effect on 3' end processing, but causes a defect in termination. To examine how Swd2 is involved in termination, we will use chromatin-immmunoprecipitation experiments to identify factors that work with or are influenced by Swd2, use genetic analysis to demonstrate the functional significance of these potential Swd2 interactors and identify novel interactors, and use mutagenesis to verify the importance of these interactions for transcription termination. ? ?
Showing the most recent 10 out of 22 publications
