The overall goal of this laboratory's research is to understand on a molecular level how the eukaryotic cell achieves its very efficient and closely regulated synthesis of rRNA. Prior work has established that the rDNA promoter is relatively large, consisting of a small proximal domain that can direct transcription, and upstream stimulatory domains that also bind transcription factors and augment the initiation process. Yet further upstream in both frog and mouse rDNA are repetitive spacer regions that serve as enhancers for transcription by polymerase I. The transcription of rRNA is catalyzed by a promoter-binding factor 'D', and a subform of polymerase I, factor 'C', that is specifically activated to participate in accurate rRNA synthesis; many cellular rRNA down-regulatory events are mediated by decreased availability of C. In the upcoming granting period we intend to purify C and D, determine how C differs from bulk pol I, and how these factors act to catalyze transcription. We will examine the basis of a number of rRNA regulatory events in which availability of C, and possibly also D, appears to mediate the control. Another major aim of the proposed studies is to understand how the frog and mouse rDNA enhancers stimulate transcription, whether they increase the number of active genes or the frequency of initiation per active gene, and the role of the functional association between the enhancer and promoter. Finally, we will introduce rRNA genes into cells in order to determine what features of the rDNA or rRNA actually serve to organize the nucleolus, the cellular organelle in which rRNA is transcribed and ribosomes are assembled.
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