Characterization of Saccharomyces cerevisiae nuclear RNase P and its RNA subunit.

In an effort to elucidate the structure and reaction mechanism of eukaryotic RNase P, the nuclear RNase P was isolated from a single cell eukaryote, Saccharomyces cerevisiae. A 369 nucleotide RNA that comigrated with RNase P activity was identified. Several possible secondary structures of this RNA were drawn using structure analysis and one of them appeared to be very similar to the phylogenetic consensus structure of eubacterial RNase P RNAs. The genomic clone of this RNA (RPR1) was obtained by screening a yeast genomic DNA library and was mapped 600 bp downstream of the URA3 gene in chromosome V. RPR1 was found to be a single copy and essential gene. A temperature sensitive RPR1 variant showed reduced in vivo RNase P activity at the nonpermissive temperature, verifying that the RPR1 RNA is the functional RNA subunit of S. cerevisiae nuclear RNase P. Northern analysis of the RPR1 RNA in a yeast RNA polymerase III temperature sensitive mutant showed that the RPR1 gene is transcribed in vivo by RNA polymerase III (pol III). In vivo complementation analyses and northern analyses of strains containing point mutations in internal promoter similarities or deletions in the upstream region of RPR1 suggested that pol III internal promoter elements are located in a putative leader sequence of the primary transcript and that the upstream sequence is necessary for efficient in vivo expression. RNase P assay with a chimeric substrate lacking a 2$\sp\prime$ hydroxyl in the nucleotide of the cleavage site suggested that the 2$\sp\prime$ hydroxyl is not necessary for RNase P reaction, unlike other RNases. By constructing a variant pre-tRNA, it was demonstrated that the single stranded structure of pre-tRNA at the cleavage site facilitates the cleavage by RNase P. The modification of either B. subtilis RNase P RNA or S. cerevisiae nuclear RNase P with several chemical reagents showed that Mg$\sp{++}$ is associated with RNase P to constitute an active enzyme and that the protein subunits of S. cerevisiae nuclear RNase P may be involved in catalysis.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105398/1/9124043.pdfDescription of 9124043.pdf : Restricted to UM users only