FUNCTION AND METABOLISM OF RNA

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The majority of the proposed program is a continuing analysis of the molecular biology of ribonuclease (RNase) P, a ribonucleoprotein enzyme that contains a catalytic RNA involved in tRNA processing. Particular focus is on the RNase P RNAs of Bacillus subtilis and Escherichia coli. The specific elements of this and the other components of the project include: 1. The continued analysis of the RNase P secondary and tertiary structure using phylogenetic structural comparisons, chemical and enzymatic structure mapping, fluorescence energy transfer distance measurements and in vitro mutagenesis. 2. The continued analysis of the RNase P RNA interaction with the RNase P protein and substrates using chemical and enzymatic footprinting, photoactivated crosslinking agents, and in vitro mutagenesis coupled with kinetic analyses. 3. The use of in vitro mutagenesis to engineer simplified versions of active RNase P RNA, thereby to define the RNA elements necessary for catalysis. 4. The analysis of the molecular complexity of RNase P in vivo. 5. The isolation and characterization of an archaebacterial RNase P. 6. The characterization of the excision of an "intron" from some Salmonella rRNAs. 7. The characterization and functional analysis of low molecular weight RNAs in B. subtilis. The program is health-related in that it will shed new light on RNA functions, including catalysis. The metabolism and many of the roles of RNA are poorly understood, yet are of fundamental significance to growth, development and disease processes, including virus proliferation.