The emerging model of eukaryotic mRNA synthesis is that transcription and mRNA processing events are carefully orchestrated in vivo by a physical association of the different machineries. For example, RNA polymerase II affects the efficiency of 3' end processing, and processing factors affect the efficiency of transcription termination downstream of poly(A) sites. We are interested in the precise molecular mechanisms involved in the coordination of these two events and have identified several new points of interaction between transcription and cleavage/polyadenylation factors. These findings suggest that the presence of processing factors at the promoter might affect the efficiency and/or specificity of transcription initiation and facilitate recycling of RNAP II back to the promoter for another round of transcription. 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. Can the activity of Ssu72 in transcription be separated from its role in 3"""""""" end cleavage? We have found that Ssu72, previously identified as a protein affecting initiation, is directly involved in mRNA 3'end cleavage. We will analyze an existing collection of ssu72 mutants to try to separate the cleavage activity of Ssu72 from a function in transcription initiation and develop new assays to help discriminate these functions. 2. What is the functional significance of the interactions of Sub1 and Ssu72 with Pta1, and how are these interactions regulated? Ssu72 and Sub1 were initially identified based on genetic interactions with TFIIB. We have found that these proteins genetically interact with the Ptal subunit of Cleavage/Polyadenylation Factor (CPF). Moreover, they physically bind Pta1 in a mutually exclusive manner. We will test the hypothesis that sequential interactions of Pta1 with Ssu72 and Sub1 are important for efficient initiation and/or cleavage of pre-mRNA. 3. Does Swd2 function in mRNA synthesis as part of CPF? This protein is intimately associated with CPF and the Set1 histone methylase. However, Swd2 depletion has no effect on 3' end processing, but causes inefficient transcription termination and reduced mRNA levels. We will test the hypothesis that Swd2 affects termination by recruiting Set1 to the transcription complex. We will identify the contact point of Swd2 with CPF and examine how disruption of this interaction affects mRNA synthesis. A genetic screen will be used to identify other important functional interactions with Swd2.
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