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

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. tuberculosis protein, 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 vitro renaturation, 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.

Insight into the role of histidine in RNR motif of protein component of RNase P of M. tuberculosis in catalysis.

RNase P, a ribonucleoprotein endoribonuclease, is involved in the 5' end processing of pre-tRNAs, with its RNA component being the catalytic subunit. It is an essential enzyme. All bacterial RNase Ps have one RNA and one protein component. A conserved RNR motif in bacterial RNase P protein components is involved in their interaction with the RNA component. In this work, we have reconstituted the RNase P of M. tuberculosis in vitro and investigated the role of a histidine in the RNR motif in its catalysis. We expressed the protein and RNA components of mycobacterial RNase P in E. coli, purified them, and reconstituted the holoenzyme in vitro. The histidine in RNR motif was mutated to alanine and asparagine by site-directed mutagenesis. The RNA component alone showed activity on pre-tRNA(ala) substrate at high magnesium concentrations. The RNA and protein components associated together to manifest catalytic activity at low magnesium concentrations. The histidine 67 in the RNR motif of M. tuberculosis RNase P protein component was found to be important for the catalytic activity and stability of the enzyme. Generally, the RNase P of M. tuberculosis functions like other bacterial enzymes. The histidine in the RNR motif of M. tuberculosis appears to be able to substitute optimally for asparagine found in the majority of the protein components of other bacterial RNase P enzymes.