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

• Published in: PloS one 2016-04, Vol.11 (4), p.e0153798-e0153798
• Main Authors: Singh, Alla, Ubaid-ullah, Shah, Ramteke, Anup K, Batra, Janendra K
• Format: Article
• Language: English
• Subjects: Arabidopsis; Bacteria; Binding sites; Biology and life sciences; Catalysis; Catalytic activity; Cloning; E coli; Enzymes; Escherichia coli; Genes; Immunology; In vivo methods and tests; Inclusion bodies; Kinetics; Laboratories; Mycobacterium; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; P protein; Physical Sciences; Protein Conformation; Protein synthesis; Proteins; Renaturation; Research and analysis methods; Ribonuclease; Ribonuclease P; Ribonuclease P - chemistry; Ribonuclease P - metabolism; Ribonucleic acid; RNA; RNA Processing, Post-Transcriptional; RNA sequencing; RNA, Bacterial - chemistry; RNA, Bacterial - metabolism; RNA, Catalytic - chemistry; RNA, Catalytic - metabolism; Serine; Substrate Specificity; Substrates; Transfer RNA; tRNA; Tuberculosis; Tuberculosis - metabolism; Tuberculosis - microbiology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0153798

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.