9304799 Kunkel Experiments in this proposal are designed to explore the mechanism of activation of human small RNA gene promoters. The primary focus of these experiments is on the human U6 gene. The transcription of vertebrate U6 genes is carried out by RNA polymerase III but the promoter is constructed of a number of DNA elements used in pol II genes. Furthermore, the U6 transcription complex is composed, in part, of factors that are shared between pol II and pol III promoters. For example, the distal region of the human U6 gene (more than 150 bp upstream of the transcriptional start site) contains a consensus octamer motif found in the same location as in the pol II-transcribed snRNA genes (e.g., U1 and U2 genes), and this element is bound by purified Oct-1 factor in vitro. Therefore, the U6 distal region is similar to the (pol II)snRNA enhancer that activates transcription by one to two orders of magnitude. The objectives of experiments proposed in this application are: 1) to map the extent of functional elements in the human U6 gene distal control region. Mutant templates will be prepared by site-directed techniques, and analyzed in vitro by gel mobility shift and DNase I footprinting assays and in vivo by transient expression in transfected human cells; 2) to isolate a transcription factor that binds to the U6 distal region whose presence has been detected already and is distinct from Oct-1 protein (Nonoct factor); 3) to characterize the step(s) in the U6 transcription initiation pathway that are controlled by activator proteins. Purified Oct-1 and Nonoct factors will be used for in vitro transcription experiments in which transcription initiation complexes are distinguished using various concentrations of Sarkosyl or after rapid separation while attached to bead-immobilized DNA templates. The relative numbers of functional transcription complexes, kinetics of complex formation and possible differential effects on reinitiation by the activator proteins will be determined; and 4) to determine whether Oct-1 protein and Nonoct factor interact or act independently to activate U6 gene transcription. The spacing between the Nonoct and octamer protein binding sites will be altered systematically, and the effect on transcriptional activation determined. Furthermore, using a DNase I footprinting assay, binding of these two factors to DNA will be investigated to determine whether cooperativity exists. %%% This research is designed to elucidate how an important class of genes are expressed at a high level in vertebrate cells. This class of genes produces the small nuclear RNAs (snRNAs) that are essential for fundamental pathways of gene expression in all higher cells. The studies will dissect the DNA sequences for one gene in this class (human U6 snRNA gene) that direct rapid RNA synthesis. Then the investigators will identify important proteins that bind to these DNA sequences and determine how these proteins stimulate efficient production of U6 snRNA. These studies will provide important information on basic mechanisms that direct how specific genes are highly expressed. ***
