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

Singh A, Ubaid-ullah S, Ramteke AK, Batra JK - PLoS ONE (2016)

Bottom Line: The protein subunit which lacks any catalytic activity, relaxes the ionic requirements for holoenzyme reaction and is indispensable for pre-tRNA cleavage in vivo.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.

View Article: PubMed Central - PubMed

Affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi -110067, India.

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.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE analysis of purified protein preparations.The two purified protein preparations of M. tuberculosis RNase P expressed in E. coli were analysed by SDS-polyacrylamide gel electrophoresis on a 14% gel. Molecular weight markers are shown in kDa.
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pone.0153798.g001: SDS-PAGE analysis of purified protein preparations.The two purified protein preparations of M. tuberculosis RNase P expressed in E. coli were analysed by SDS-polyacrylamide gel electrophoresis on a 14% gel. Molecular weight markers are shown in kDa.

Mentions: The DNA encoding RNase P protein component, cloned in expression vector pVex11, was expressed in E. coli BL21 (λDE3) cells. The expressed protein was localized in the inclusion bodies. 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). The holoenzymes obtained by reconstitution of two proteins with RNase P RNA are termed as RNase P-U holoenzyme and RNase P-G holoenzyme.


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

Singh A, Ubaid-ullah S, Ramteke AK, Batra JK - PLoS ONE (2016)

SDS-PAGE analysis of purified protein preparations.The two purified protein preparations of M. tuberculosis RNase P expressed in E. coli were analysed by SDS-polyacrylamide gel electrophoresis on a 14% gel. Molecular weight markers are shown in kDa.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835064&req=5

pone.0153798.g001: SDS-PAGE analysis of purified protein preparations.The two purified protein preparations of M. tuberculosis RNase P expressed in E. coli were analysed by SDS-polyacrylamide gel electrophoresis on a 14% gel. Molecular weight markers are shown in kDa.
Mentions: The DNA encoding RNase P protein component, cloned in expression vector pVex11, was expressed in E. coli BL21 (λDE3) cells. The expressed protein was localized in the inclusion bodies. 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). The holoenzymes obtained by reconstitution of two proteins with RNase P RNA are termed as RNase P-U holoenzyme and RNase P-G holoenzyme.

Bottom Line: The protein subunit which lacks any catalytic activity, relaxes the ionic requirements for holoenzyme reaction and is indispensable for pre-tRNA cleavage in vivo.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.

View Article: PubMed Central - PubMed

Affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi -110067, India.

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.

No MeSH data available.


Related in: MedlinePlus