Understanding the mechanisms of transcription of the genes that code for 45S pre-ribosomal RNA is essential if we are to understand both normal and abnormal growth processes, e.g. wound healing and neoplasia. Ribosome biogenesis, and therefore the expression of the ribosomal RNA genes, is coordinated with the rate of cell growth, and responds to a variety of signals, depending upon the cell type studied. The long-term objective of our research is to determine the mechanism(s) by which ribosomal gene transcription is regulated. The promoters of vertebrate ribosomal RNA genes (rDNA) have analogous functional elements, although their nucleotide sequences are often quite different. Further, there are both conserved, and homologous but species-specific trans-acting factors, as well as species-specific patterns of utilization of these homologous factors. The mammalian RDNA promoters consist of two broadly defined elements: a core promoter element (CPE) and an upstream promoter element (UPE). The CPE is necessary and sufficient for transcription in vitro, and necessary for transcription in vivo. However, the UPE is required for transcription in vivo, and for efficient transcription in vitro, as well as for the formation of a stable preinitiation complex. At least two trans-acting factors are required for accurate and efficient transcription by RNA polymerase I, SL-l and UBF. SL-l is required for transcription, while UBF activates transcription. We have purified rat SL-l, the factor required for species-specific transcription, and determined that it interacts both the CPE and the UPE. We have purified and cloned rat UBF. Purified rat UBF, which consists of two polypeptides, footprints between -120 and -50 of the rat rDNA promoter, within the domain defined as the UPE. However, for UBF to affect transcription in vitro, the binding site for SL-l in the UPE must be intact. Moreover, we have found that the two polypeptides present in preparations of UBF are probably the products of two separate mRNAs, and that they are closely related proteins with what may be significant differences in DNA-binding or function. This hypothesis will be pursued. Rat SL-l will be purified and characterized, and the specific roles of UBF and SL-l in transcription will be defined. Further, our results indicate that at least one other factor is required for the formation of a stable preinitiation complex, and we will purify that factor. By combining in vitro transcription and DNA-binding assays using highly purified transcription factors and various mutants of the promoter with in vivo expression assays, we will determine the mechanism by which transcription is accomplished. The proposed experiments will increase our understanding of the mechanism of transcription of this multi-gene family, and the mechanism by which its transcription is regulated.
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