5S RNA and ribosomal RNA transcription rates, and expression of ribosomal proteins are closely tied to cellular growth rate in order to balance elaboration of ribosomes. This proposal is aimed at studying the mechanism of coordinating expression from these genes, which are transcribed by distinct RNA polymerases. In Acanthamoeba, rRNA transcription is regulated by polymerase I modification, and recent results show that 5S RNA transcriptional shutdown is accompanied by a loss in TFIIlA transcriptional and DNA binding activity in nuclei. The mechanism of this loss is unknown. This study will examine the change in TFIIlA activity by several approaches: The level of TFIIlA protein present in the whole cell will be revealed by Western blot analysis. A change would indicate that there is less TFIIlA in the cell. If so, an investigation of the mechanism for this change will involve cloning the gene for TFIIlA and determining whether TFIIlA mRNA declines in parallel with TFIIlA protein. If so, nuclear run off experiments will determine whether the TFIIlA mRNA level is regulated transcriptionally. If TFIIlA is regulated transcriptionally, an investigation into the mechanism of this regulation will be carried out. First, the promoter of the cloned TFIIlA gene will be dissected using in vitro transcription. If TFIIlA mRNA levels are not regulated transcriptionally, TFIIlA mRNA association with polysomes will be examined to determine if translation is specifically hindered. Alternatively, if the overall cellular level of TFIIlA remains constant, the cellular localization of TFIIlA, eg. as a complex with 5S RNA in the cytoplasm, will be investigated by analyzing the distribution of TFIIlA in nuclei and the cytoplasm, and by determining the amount of 7S RNP and 42S RNP in the cytoplasm at different stages of cellular development and 5S RNA transcriptional activity. If the amount of TFIIIA protein present in nuclei is constant, but only the DNA binding activity of the factor is altered, then TFIIIA must be modified so that it cannot interact with the 5S RNA gene. An investigation of modification will be carried out by structural comparison of TFIIlA from active and inactive cells. Later in Acanthamoeba development, the activity level of TFIIIC is reduced, presumably to shut down other type 2 polymerase III transcription. We will investigate the mechanism of this change using an approach similar to the above study of TFIIIA.
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