In eucaryotes, transcription is carried out by three evolutionary related RNA polymerases, each of which recognizes a different type of promoter. The U2 and U6 snRNA genes are transcribed by RNA polymerase II and III, respectively, yet the U2 and U6 promoters are much more similar to each other in structure than they are to either the RNA polymerase II promoters of mRNA genes or the internal RNA polymerase III promoters of the tRNA and 5S genes. They consist of a proximal sequence element, the PSE, located around -50, and an enhancer or distal sequence element (DSE) located around -200. The U6 promoter contains in addition an A/T rich element at -25 which determines its RNA polymerase III specificity. Amazingly, the U6 A/T rich element can direct the assembly of an RNA polymerase III initiation complex by binding TFIID, the TATA-box binding protein (TBP) that directs the assembly of RNA polymerase II initiation complexes on the TATA boxes of mRNA promoters. Little is known about the RNA polymerase II U2 transcription complex; however, it is different from transcription complexes assembled on mRNA promoters because it has different elongation properties. Indeed, the human RNA polymerase II snRNA genes contain a termination signal, the 3' box, which is recognized only by transcription complexes derived from RNA polymerase II snRNA promoters. Thus, if the U2 promoter is replaced by a mRNA promoter such as the beta-globin promoter, RNA polymerase II ignores the 3' box and fails to terminate transcription. The biochemical basis for this observation is unknown. This application describes the continuation of our analysis of the RNA polymerase III U6 transcription complex and the undertaking of a similar analysis of the RNA polymerase II U2 transcription complex. The general goals are to compare the two types of transcription complexes and thus provide an understanding of how RNA polymerase specificity is achieved, as well as to examine the mechanisms that govern transcription termination in the RNA polymerase II snRNA genes. First, we will identify and purify the factor that functionally recognizes the U6 PSE, and isolate a cDNA encoding it. Second, we will purify other components of the U6 transcription complex, in particular TFIIIB and RNA polymerase III, so as to be able to pursue functional reconstitution studies and examine the assembly pathway of the U6 initiation complex. Third, we will characterize the native form on TBP involved in RNA polymerase III transcription and determine whether TBP-associated factors are required for trans-activation of basal U6 transcription by Oct-1. In the fourth specific aim, we will establish the assembly pathway of the U6 initiation complex and define the domains of TBP and the PSE-binding factor required for this process. In the last specific aim, we will establish a fractionated in vitro system for transcription of the RNA polymerase II snRNA genes, determine which of the transcription factors involved in U6 transcription by RNA polymerase III are also involved in U2 transcription by RNA polymerase II, and identify factors required for transcription termination at the 3' box. Together, these studies will further our understanding of fundamental transcription mechanisms.
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