Effect of overproduction of C5 protein on biosynthesis of M1 RNA in escherichia coli

Abstract

RNase P, the enzyme responsible for the biosynthesis of the 5``-termini of mature tRNA molecules in Escherichia coli, is composed of an RNA subunit (M1 RNA) coded by rnpB and a protein subunit (C5 protein) coded by rnpA. While M1 RNA can, by itself, carry out the catalytic reaction in vitro, both components are essential for the activity of RNase P in vivo. The accurate function of the C5 protein in vivo is unknown, but it has been assumed that M1 RNA biosynthesis depends on the synthesis of C5 protein. Using plasmids that could overproduce C5 protein upon induction with IPTG, we investigated effects of C5 protein on M1 RNA biosynthesis. To construct the plasmids, the rnpA gene was fused to the tac promoter of expression vector pKK223-3. About 40 bp DNA sequences coding the N-terminal region of C5 protein were chemically synthesized to keep the optimum distance between the S/D sequence of the vector and the translation start of the rnpA and to choose efficient codons. The plasmids also harbored the rnpB gene with the internal deletion which allowed us to directly examine M1 RNA transcription by analyzing metabolically unstable M1 RNA transcripts derived from the plasmids. The tac promoter expression system of the plasmids produced a high level of C5 protein upon induction with IPTG, but the overexpressed C5 protein had no significant effect on the M1 RNA transcription from plasmids.