Regulation of RNase P RNA biogenesis

Abstract

The ribonucleoprotein enzyme RNase P was initially characterized as a tRNA-processing enzyme that removes extraneous 5´ sequences from precursor tRNAs to generate mature 5´ termini. Escherichia coli RNase P is composed of a large RNA subunit (M1 RNA) and a small protein subunit (C5 protein). In this thesis, first, it was investigated whether C5 protein plays a role in maintaining metabolic stability of M1 RNA. The sequestration of C5 protein available for M1 RNA binding reduced M1 RNA stability in vivo, and its reduced stability was recovered via overexpression of C5 protein. In addition, M1 RNA was rapidly degraded in a temperature-sensitive C5 protein mutant strain at nonpermissive temperatures. Collectively, the results demonstrate that the C5 protein metabolically stabilizes M1 RNA in the cell. Analysis with various RNase mutants strains suggests that RNase PH is involved in the turnover of free M1 RNA. Cis-acting elements that might affect RNA stability were also investigated. For this purpose, M1 RNA, which is a highly stable RNA, was used as a model system. To find motifs essential for M1 RNA stability, M1 RNA derivatives lacking specific motifs or mutated were constructed and analyzed for their in vivo stabilities. The results suggest that the P1 stem structure is the most important element for maintaining M1 RNA stability, and then the P14 and P17 stem-loops are the next. Since both subunits are assembled in a 1:1 ratio, expression of M1 RNA and C5 protein should be coordinately regulated for RNase P to be efficiently synthesized in the cell. However, it is not known yet how the coordination occurs. In this thesis, it was investigated how overexpression of C5 protein affects expression of the rnpB gene encoding M1 RNA, using a lysogenic strain, which carries an rnpB-lacZ transcription fusion. Primer extension analysis of rnpB-lacZ fusion transcripts showed that the overexpression of C5 protein increased the amount of the fusion transcripts, sugges...