My long-term goal is to understand how the eukaryotic nucleus function in gene expression. Because the nucleolus is the most clearly defined nuclear region and the location of pre-rRNA synthesis and processing, accounting for about half of cellular transcription and three quarters of steady state RNA, we are focusing on the nucleolus and rRNA production. Much progress has been made, by many labs including our own, on the basal rDNA transcription process, but much less is known about how the rDNA enhancer function and still less is known about what organizes the nucleolus an various nuclear domains. We propose to addresses these issues; [1] During the current funding, we found that plasmids transiently introduced into cultured cells show a very selective targeting,, depending what sequences they carry; the rDNA promoter, or enhancer, or terminator targets its host plasmid (greater than 50,000 copies/transfected cell) specifically to the nucleolus; a pol II promoter targets to nucleoplasmic sits (evidently """"""""speckles""""""""); and pol III promoters target to yet different sites, perinucleolar foci. This targeting is not dependent on transcription of the plasmid, and only lesser than 0.1% of the targeted pol I promoter-containing plasmids are transcribed. Experiments are proposed to learn more about this targeting, including improving the detection and the mode of DNA introduction, better defining the sequences that direct each of the three kinds of targeting, learning about the cellular components that """"""""anchor"""""""" the transfected plasmids to their different targeting sties and what these sites represent in the cell, and discerning the mode of DNA entry. [2] During the current period of funding we have also become able to reproduce the action of the rDNA enhancer in vitro. Surprisingly, it is not a true stimulator, but it functions to abrogate the action of an inhibitor that is activated upon incubation of the template complex and ATP. Much of second part of this proposal concerns elucidating the action of this inhibitor and if the enhancer in preventing this inhibition. Experiments are designed to learn when in the transcription process the inhibitor and the enhancer act, whether this may be related to the conversion of factor C* from its initiation to elongation mode, and to discern what the inhibitor is. In related experiments, we will pursue our recent success at showing that the rDNA enhancer acts to make more genes transcribed, not to alter the transcription rate on each active gene, by observing individual transcription units; we will now use this analysis on selected pol II enhancers, to learn how they function at the level of the individual reinitiating gene. Through these studies we hope to get a better understanding about the action of transcriptional enhancers and about the interactions that serve to differentiate and organize the different domains of the eukaryotic nucleus.
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