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, which comprises over 75 percent of the total cellular RNA. In the previous years of funding on GM27720, the investigator demonstrated systems for in vitro and in vivo expression of Xenopus and mouse rDNA, localized the promoter sequences, and studied the cellular components responsible for this expression and its regulation. The investigator now wishes to extend these studies, and to that end have proposed four Specific Aims. First, the investigator will purify and characterize the rDNA transcription factors, focusing initially on D (which in conjunction with upstream binding factor (UBF) selectively binds the rDNA promoter) and C* (the recently resolves component that evidently confers regulated specific initiation-competence on the polymerase). The investigator describes a number of studies to better understand how D and C*, as well as UBF and polymerase I, and a less well-characterized component (ST1), function.
The second aim will be to examine the action of upstream rDNA transcriptional elements: the frog 60/81 bp repeats and 0/1 repeats, the mouse 140 bp repeats and the mouse spacer promoter region. The investigator proposes experiments to determine whether the enhancer repeats function via chromatin assembly, whether they affect the number or activity of active rRNA genes, and their factor binding and helical requirements. In the third Aim, the investigator will extend the studies of the frog rDNA promoter, examining its internal spacing and sequencing requirements, its ability to function cross-species, and especially the basis of the selective transcription (that occurs under certain conditions) of an rRNA gene deleted of all known upstream specificity sequences.
The fourth Aim addresses the organization of the rDNA transcription unit. The investigator will examine the (apparently) altered chromatin structure of active rRNA genes and the sensitivity of this polymerase I transcription to topoisomerase I inhibition. Finally, the investigators will determine whether """"""""terminal knobs"""""""", a hallmark of rRNA transcription units in Miller spreads, are due to primary processing complexes assembled on the nascent transcripts.
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