Mechanisms of transcription and regulation of expression of stable RNAs (precursor ribosomal and 5S) are being studied. Evidence for regulation of stable RNA synthesis by modulation of auxilliary transcriptional protein levels, by RNA polymerase modification and by trans-acting proteins has been obtained in this lab and others. As an initial step in studying the mechanism of regulation of 5S RNA transcription, cloning of the 5S RNA gene, determination of its overall genomic structure and primary sequence is proposed. Development of a cell-free transcription system for 5S RNA to complement the rRNA transcription system already in use in this lab is also proposed. The 5S RNA transcription system will be utilized to examine the molecular mechanism of developmentally regulated shutoff of 5S RNA expression. Emphasis will be on studying the relationship between the mechanisms of the parallel shutdown of 5S and ribosomal RNA resulting from cessation of cellular growth and proliferation. Studies of the structure of the rRNA promoter are ongoing. Deletion mutations constructed and tested for transcriptional activity in vitro have identified a 50 base pair region proximal to the rRNA transcription start site as the promoter. This proposal aims to produce a series of point mutations within this sequence in order to more precisely define the interactions between the promoter and auxilliary transcriptional proteins.
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