REGULATION OF EUKARYOTIC RIBOSOMAL RNA TRANSCRIPTION

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Our main objective is to identify, isolate, and determine the function of components in the ribosomal RNA (rRNA) transcription system, so that the regulatory mechanisms that govern RNA synthesis can be manipulated and analyzed at the biochemical level. We had previously developed a fractionated in vitro system that accurately initiates RNA synthesis from the human ribosomal promoter, and we had defined the cis regulatory sequences that specify promoter function. In addition, we have recently identified and partially purified a species-specific promoter recognition factor, called SLl. Most importantly, preliminary DNA binding studies and site-directed mutagenesis analysis suggests that SLl, in conjunction with another transcriptional component, possibly RNA polymerase I itself, forms a complex that can then recognize and interact with the upstream control element of the promoter. In this renewal grant, we propose to analyze the nature and specificity of the protein-DNA and protein-protein interactions that occur between SL1,RNA polymerase I and promoter sequences. These biochemical studies should provide us with new insights concerning the molecular mechanism that mediates transcriptional activation of mammalian rRNA genes. First, a variety of DNA binding experiments, including DNAse footprinting, Carothers gel binding assays, DMS methylation protection, MPE protection, and alkylation-interference using both wild-type and mutant templates will be used to analyze the protein-DNA interactions that direct promoter recognition. Next, we plan to purify SLl as well as other proteins that form a complex with SLl and raise monoclonal antibodies directed against these transcription factors. If high titer antibodies can be obtained, we will then use them to help us identify and characterize the SLl polypeptide as well as its interaction with other polypeptides involved in the transcription initiation reaction. The antibodies can also be used to detect and quantitate the presence of transcription factors in the nucleolus of cells under different growth and environmental stress conditions in order to correlate transcriptional activation with the presence of specifc trans-activating factors. After obtaining highly purified and active SLl, we will attempt to microinject the purified factor into a variety of cell types such as human-mouse chimeras, to test the idea that SLl may be responsible for nucleolar dominance in mammalian-cell hybrids. Finally, we will attempt to clone and characterize the genes encoding SLl and other relevant transcription factors with the aim of eventually developing expression vectors that can overproduce one or more of these low abundance transcription proteins.