NMR study on the escherichia coli ribonuclease P protein

Abstract

Ribonuclease P is an enzyme which releases the 5’-precursor sequence from immature tRNAs and produces mature tRNAs. RNase P consists of a catalytic RNA subunit and a cofactor protein subunit, and especially for Escherichia coli, RNase P is composed of M1 RNA (377nt) and C5 protein (119aa). Although in vitro data shows that M1 RNA has catalytic activity at high concentration of magnesium ion without the C5 protein, in vivo data indicate that C5 protein is essential and seems to affect the catalytic activity of RNase P. The accurate function of the C5 protein in vivo is unknown, but three dimensional structure of C5 protein could help to understand the structural and functional role of C5 protein. Structural studies by NMR spectroscopy require millimolar concentration of protein that has not been achieved successfully so far. In this study, $^{13}C$ and $^{15}N$ labeled C5 protein with about 1 mM concentration was obtained by ammonium sulfate precipitation and ion-exchange chromatography. Partial assignments of the backbone resonances ($C^α, C^β, CO, N^H, and H^N$) of C5 protein were performed using a combination of 2D or 3D triple resonance NMR experiments; CBCA(CO)NH, HNCACB, $^{1}H-^{15}N$ HSQC, and HNCO.