Mutational analyses of yeast nuclear RNase P.

Abstract

Ribonuclease P (RNase P) is an ubiquitous endoribonuclease that cleaves precursor transfer RNA molecules to form mature 5$\sp\prime$ termini. Unlike other ribozymes, it does not recognize its substrates by base-pairing but rather by recognition of conserved tertiary structures in the tRNAs. RNase P is a metalloenzyme, requiring magnesium ions as cofactors for catalysis. In both prokaryotes and eukaryotes the enzyme exists as a ribonucleoprotein, but the eubacterial RNA moieties are catalytic alone in vitro, demonstrating that the RNA is responsible far substrate recognition and catalysis. This dissertation investigates the functional contributions of structural elements of the RNA subunit of yeast nuclear RNase P. Using the yeast Saccharomyces cerevisiae as model system, mutagenesis techniques and in vivo complementation were used to identify regions of the RNA that are essential for function in vivo. Previous phylogenetic studies revealed a high conservation of secondary structure in yeast RNase P RNAs. To correlate this observed conservation with function, in vivo complementation experiments were performed using heterologous yeast RNase P RNAs. The results identified sequences and structures in the RNA that are not essential for interaction with species-specific proteins, processing or localization, and suggested other positions that may be candidates for such processes. Based on these observations, a series of highly conserved sequences and structures that could have important functional roles were analyzed by directed mutagenesis. Results from deletion experiments showed good correlation with our predictions since structures which sequences are not conserved proved not to be essential for function. Highly conserved nucleotides that are invariant between eubacterial and yeast RNase P were also investigated by randomization mutagenesis. Some mutations gave conditional growth mutants. These were used to study the effects of the mutations in different aspects of RNase P function. Through partial purification of mutant holoenzymes it was also possible to inspect enzyme function in vitro. Kinetic analyses have revealed that a highly conserved subdomain in the RNA is involved in catalysis, possibly by coordinating magnesium ion cofactors at the cleavage site.