We use the human snRNA genes to study fundamental mechanisms of transcription. They represent an excellent model system for this purpose for a number of reasons. First, snRNA genes contain two types of promoters, one type recognized by RNA polymerase (pol) II and one type recognized by pol III. The pol II and III promoters are very similar in structure and indeed they recruit several common transcription factors. Thus, any difference in the initiation complexes assembled on the two types of promoters is likely to be relevant for the determination of RNA polymerase specificity. Second, these promoters are relatively simple. The core pol II snRNA promoters such as the U1 promoter consist of just one essential element, the proximal sequence element or PSE, which recruits a multisubunit factor called SNAPc. The core pol III snRNA promoters such as the U6 promoter contain a PSE as well as a TATA box, which recruits the TATA box binding protein TBP. Both the pol II and III snRNA promoters contain a distal sequence element or DSE, which activates transcription and contains an octamer sequence that recruits the POU domain protein Oct-1 and a so-called SPH site that recruits the zinc finger protein Staf(ZNF143). Third, we have recently identified a set of well-defined factors required and sufficient for transcription from a pol III snRNA promoter. The identification of this basal pol III transcription machinery allows us to study how it is regulated, and indeed we have shown that CK2 can both activate and repress transcription by phosphorylating different targets within this machinery. The identification of this basal pol lll transcription machinery allows us to study how it is regulated. We propose to 1) continue characterizing the mechanisms of U6 transcription including the architecture of SNAPc, the assembly of the U6 transcription initiation complex, and the impact of Oct-1 on the assembly of this initiation complex; 2) characterize how ZNF143 and Oct-1 cooperate to activate U6 transcription from chromatin templates; and 3) determine how and when CK2 regulates pol III transcription from the human U6 promoter. Together, these experiments will reveal how the U6 transcription complex assembles and specifically recruits pol Ill, and how the assembly and activity of the complex is regulated by activators such as Oct-1 and ZNF143, and by kinases such as CK2, during cell growth and proliferation.
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