title: Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function.
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. 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.
authors.affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi -110067, India.
authors.forename: Alla
authors.initials: A
authors.lastname: Singh
authors.affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi -110067, India.
authors.forename: Shah
authors.initials: S
authors.lastname: Ubaid-ullah
authors.affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi -110067, India.
authors.forename: Anup K
authors.initials: AK
authors.lastname: Ramteke
authors.affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi -110067, India.
authors.forename: Janendra K
authors.initials: JK
authors.lastname: Batra
mesh_terms: D002384:Catalysis; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014376:Tuberculosis / Q000378:metabolism* / Q000382:microbiology
chemical_list: D012329:RNA, Bacterial; D016337:RNA, Catalytic; D043262:Ribonuclease P