Studies on the roles of C5 protein, the protein component of Escherichia coli RNase P

Abstract

RNase P is an endonuclease which cleaves 5′ extra sequences from precursors of tRNAs to generate the mature 5′ ends of tRNAs. E. coli RNase P consists of two subunits, a large RNA (M1 RNA) and a small basic protein (C5 protein). Since M1 RNA alone can cleave its substrates at the correct position at high $Mg^{2+}$ concentration in vitro, it is the catalytic subunit. However, M1 RNA needs C5 protein for efficient catalysis at physiological $Mg^{2+}$ concentrations. Furthermore both M1 RNA and C5 protein are essential for the activity of RNase P in vivo. C5 protein is known to play a critical role in recognition and binding of some substrates specifically. To examine the role of C5 protein in the assembly of the holoenzyme and the catalysis by the holoenzyme, deletion derivatives of C5 protein were constructed as soluble MBP (maltose binding protein) fusion protein. The C5 protein, with large terminal deletions, retained its promoting activity of RNase P catalysis under protein excess conditions in vitro. Some deletion derivatives complemented the temperature sensitive phenotype of E. coli A49 cells carrying the rnpA49 mutation. This complementation ability also suggests that part of the C5 protein is enough to produce the catalytic activity of RNase P in vivo. Both the central conserved region, called the RNR motif, and the C-terminal region are essential for the binding of C5 protein to M1 RNA. Meanwhile, the N-terminal region contributes to promoting RNase P catalysis in ways other than binding to M1 RNA. Although C5 protein was originally identified as the protein component of RNase P, it is possible that C5 protein participates in other cellular metabolisms. An RNA molecule capable of being cleaved by RNase P was isolated from the pool of RNA aptamers binding to C5 protein which were selected by SELEX (systematic evolution of ligands by exponential enrichment) through the 13th round from an E. coli genomic RNA library carrying an overlapping set of inserts o...