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.
Highlights
In a cell, transfer RNA acts as an adaptor between the coding sequence in messenger RNA and amino acid sequence in proteins [1]
The protein component of RNase P of M. tuberculosis was further purified from inclusion bodies by two different methods resulting in two protein preparations termed RNase P-U and RNase P-G protein, respectively (Fig 1)
RNase P has been shown to be essential for survival of M. tuberculosis [26]
Summary
Transfer RNA (tRNA) acts as an adaptor between the coding sequence in messenger RNA and amino acid sequence in proteins [1]. TRNA molecules have extra sequences at 5’ and 3’ ends, referred to as 5’ leader and 3’ trailer, respectively. The tRNA along with its extra sequences is denoted as precursor tRNA (pretRNA) [3] These extra sequences in the pre-tRNA must be removed to form a mature tRNA product that can attain correct conformational shape and take part in the protein synthesis process. This process of removal of extra sequences is referred to as pre-tRNA processing. The 3’ trailer sequence is processed by enzymes like RNase D, E, F, Z, etc. The 5’ leader sequence is removed by a single endonuclease called RNase P [7]
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