Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function

July 28, 2017

Title

Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function

Author

Alla Singh, Shah Ubaid-ullah, Anup K. Ramteke, Janendra K Batra

Year

2016

Journal

PLoS ONE

Abstract

RNase P is an essential enzyme that processes 5' end leader sequence of pre-tRNA to generate mature tRNA. The bacterial RNase Ps contain a RNA subunit and one protein subunit, where the RNA subunit contains the catalytic activity. The protein subunit which lacks any catalytic activity, relaxes the ionic requirements for holoenzyme reaction and is indispensable for pre-tRNA cleavage in vivo. In the current study, we reconstituted the M.tuberculosis RNase P holoenzyme in vitro. We prepared the RNase P protein through two different strategies that differ in the conditions under which the recombinant M. tuberculosisprotein, expressed in E. coli was purified. The mycobacterial RNase P protein which was purified under native conditions subsequent to isolation from inclusion bodies and in vitrorenaturation, was capable of cleaving pre-tRNA specifically without the requirement of RNase P RNA. However, the preparation that was purified under denaturing conditions and refolded subsequently lacked any inherent pre-tRNA processing activity and cleaved the substrate only as a component of the holoenzyme with the RNA subunit. We found that the two RNase P protein preparations attained alternative conformations and differed with respect to their stability as well.

Instrument

J-815

Keywords

Circular dichroism, Secondary structure, Tertiary structure, Protein denaturation, Thermal stability, Biochemistry