Abstract
Ribonuclease P (RNase P) is an essential RNA enzyme found in all phylogenetic domains that is best known for catalyzing the 5’ endonucleolytic cleavage of precursor transfer RNAs (pre-tRNAs). In bacteria, the enzyme consists of a single, catalytic RNA subunit and one small protein, while the archaeal and eukaryotic enzymes have 4-10 proteins in addition to a similar RNA subunit. The RNA has been shown to act as a ribozyme at high salt in vitro; however the added protein optimizes kinetics and makes specific contacts with the pre-tRNA substrate. The bacterial protein subunit also appears to be required for the processing of non-tRNA substrates by broadening substrate recognition tolerance. In addition, the immense increase in protein content in the eukaryotic enzyme suggests substantially enlarged capacity for recognition of additional substrates. Recently, intron-encoded box C/D snoRNAs and HRA1 RNA were shown to be likely substrates for yeast RNase P. In addition, yeast RNase P seems to be inhibited by single stranded RNA. This inhibition was shown to be specific to S.cerevisiae RNase P but not bacterial RNase P with poly-ribonucleic acid homopolymers having varying levels of inhibition (polyG>polyU>>poly A>>>polyC). This inhibition is also size dependent with larger RNA being inhibitory. In addition, the sequence specificity seen with the small homoribopolymers is lost, as larger RNA of various sequences show relatively high levels of inhibition. To further characterize the extent of RNase P inhibition by these various inhibitors, the location of the inhibitor contact is being mapped by crosslinking. Individually 6xHis tagged strains are being used to enable isolation of crosslinks induced by UV light. The characterization of the eukaryotic specific inhibitor interaction with yeast RNase P will provide further information about the evolution of the essential activity of tRNA processing.
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