In eukaryotic cells, the promoter for the ribosomal RNA gene consists of several regions: a core and a varying number of upstream elements. The role of the core is to bind one or more trans-acting transcription factors. In Acanthamoeba, a single factor, TIF, binds to a defined sequence in the core. In this laboratory, TIF has been very highly purified, and its interaction with the DNA has been characterized in detail by mutagenesis of the promoter and footprinting techniques. Interactions between TIF and polymerase also have been characterized by the same methods. Among the significant findings are that TIF directs RNA polymerase I to bind over the transcription start site by protein-protein contact; polymerase makes no sequence-specific contacts with the DNA during binding. Further, rRNA transcription is regulated by modification of the RNA polymerase, and the modification prevents polymerase from binding to the promoter suggesting a possible effect on the TIF-polymerase interaction. In order to more fully understand the mechanism of initiation and regulation of rRNA expression, the cloning of the TIF gene is proposed. The purified TIF will be partially sequenced and an oligonucleotide probe synthesized from the predicted DNA sequence. A genomic library, which because of the small genomic complexity of Acanthamoeba consists of only 9200 plaques, will be screened for the TIF gene. Following sequencing, the genomic clone will be used to screen a cDNA library. The cloned TIF gene will be mutagenized, transcribed and translated in vitro, and the functional domains of the protein identified using several assays. Template commitment, footprinting, and gel shift assays will be used to evaluate DNA binding. Transcription run-off assays and DNA footprinting will be used to identify TIF-mediated polymerase binding and activation. Footprinting assays will determine the effects of mutant TIF binding on DNA conformational changes associated with the wild-type factor binding. In the long term, the details of the interaction of TIF and polymerase will be evaluated using site-specific point mutagenesis of the TIF gene and cloned RNA polymerase subunit gene(s).
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