In addition to its serving as a model system for studies of transcription mechanisms, Acanthamoeba is the causative agent of amoebic keratitis, a serious and currently difficult to treat corneal infection associated with contact lens wear. This is a proposal to continue studies of the molecular biology of this agent, specifically, the mechanism of initiation and regulation of eukaryotic 5S RNA transcription. Acanthamoeba castellanii, is an excellent model for the study of eukaryotic transcription using biochemical approaches, and significant findings during the last funding period lead logically into the proposed experiments. The applicant has cloned the 5S RNA gene from Acanthamoeba, and analyzed its organizational structure and sequence. The cloned gene sequence matches the sequence of authentic Acanthamoeba 5S RNA, which the applicant had sequenced earlier, and is transcribed in vitro, showing that it is a true gene and not a pseudogene. 5S RNA transcription is down regulated in response to cessation of growth and encystment of Acanthamoeba, a response which parallels down regulation of large rRNA precursor in Acanthamoeba nd other eukaryotic cells. The experiments proposed are aimed at an examination of the mechanism of coordinate regulation of these two transcription units. Three categories of experiments are proposed: (1) Purification and characterization of the trans-acting protein factors required for transcription of the 5S RNA gene. In eukaryotes, there is some controversy concerning how many separate transcription factors are needed for 5S RNA and tRNA initiation complex formation, so this will be a topic of study. (2) Studies of the mechanism of initiation of 5S RNA transcription. Relatively few completely homologous in vitro transcription systems exist for eukaryotic 5S RNA. The Acanthamoeba system is one of them. This laboratory has extensively studied the mechanism of large rRNA transcription, and have made important discoveries concerning the fundamental mechanisms of initiation. A single transcription factor binds to an upstream site on the gene and directs, by protein-protein interaction, RNA polymerase I to the transcription start site. Recently, results from another laboratory have suggested that an exactly homologous mechanism applies to 5S RNA and tRNA transcription by RNA polymerase III, but this must be repeated in other eukaryotic systems for verification.
The aim of the studies proposed is to confirm or disprove that this is the universal mechanism of eukaryotic transcription initiation. Analysis of promoter sequences, especially those believed to interact with the central transcription factor are planned. The mechanism by which the ancillary """"""""assembly"""""""" factors load the fundamental factor onto its site, and how (and if) binding affects the conformation of the DNA so as to stimulate various steps in the initiation process are topics for study. (3) Study the mechanism of coordinate regulation of 5S RNA and rRNA transcription. Earlier studies from this laboratory demonstrated that large rRNA transcription is regulated by modification of polymerase I so that it no longer interacts with the factor bound to the promoter. Polymerases I and III share a subunit which is modified in both during transcriptional shutdown, so coordinate regulation by co-modification is possible. However, mechanisms involving alteration of transcription factor levels or activities will also be examined, as well as mechanisms involving relocation of factors into non nuclear cellular compartments.
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