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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

Pre-tRNA processing by components of RNase P upon treatment with micrococcal nuclease and proteinase K.RNase P-G protein, RNase P-U protein and RNase P RNA were treated with either micrococcal nuclease (MN) or proteinase K (PK), followed by activity assay using standard conditions. N, untreated sample; MN, treated with micrococcal nuclease; PK, treated with proteinase K; H-G and H-U refer to holoenzymes reconstituted with RNase P-G and RNase P-U proteins, respectively.
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pone.0153798.g005: Pre-tRNA processing by components of RNase P upon treatment with micrococcal nuclease and proteinase K.RNase P-G protein, RNase P-U protein and RNase P RNA were treated with either micrococcal nuclease (MN) or proteinase K (PK), followed by activity assay using standard conditions. N, untreated sample; MN, treated with micrococcal nuclease; PK, treated with proteinase K; H-G and H-U refer to holoenzymes reconstituted with RNase P-G and RNase P-U proteins, respectively.

Mentions: The RNase P components, RNase P-G and RNase P-U proteins, and RNase P RNA were separately treated with micrococcal nuclease to determine if the protein preparation contained any contaminating RNA component of E. coli. After the treatment, micrococcal nuclease was inactivated by addition of EGTA to the reaction as it requires calcium ions for activity. The components were separately treated with proteinase K also to confirm that the activity seen with RNase P-G protein was within the protein. After treatment with nuclease and proteinase, the treated components were used to assay the pre-tRNA processing activity using standard conditions. The activity of RNase P-G protein alone was abolished upon treatment with proteinase K, however it was unaffected after treatment with micrococcal nuclease (Fig 5). As seen before, RNase P-U protein did not show any activity by itself under any conditions (Fig 5). The RNase P RNA alone activity was abolished upon treatment with micrococcal nuclease, and remained unaffected after treatment with proteinase K (Fig 5). When the holoenzymes, reconstituted with RNA component and RNase P-G and PU proteins were treated with proteinase K, their activities were lost (Fig 5). However, treatment of holoenzymes with nuclease resulted in the loss activity of that with P-U protein, whereas there was activity in RNase P-G holoenzyme confirming it to be the activity of RNase P-G protein alone (Fig 5).


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)

Pre-tRNA processing by components of RNase P upon treatment with micrococcal nuclease and proteinase K.RNase P-G protein, RNase P-U protein and RNase P RNA were treated with either micrococcal nuclease (MN) or proteinase K (PK), followed by activity assay using standard conditions. N, untreated sample; MN, treated with micrococcal nuclease; PK, treated with proteinase K; H-G and H-U refer to holoenzymes reconstituted with RNase P-G and RNase P-U proteins, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153798.g005: Pre-tRNA processing by components of RNase P upon treatment with micrococcal nuclease and proteinase K.RNase P-G protein, RNase P-U protein and RNase P RNA were treated with either micrococcal nuclease (MN) or proteinase K (PK), followed by activity assay using standard conditions. N, untreated sample; MN, treated with micrococcal nuclease; PK, treated with proteinase K; H-G and H-U refer to holoenzymes reconstituted with RNase P-G and RNase P-U proteins, respectively.
Mentions: The RNase P components, RNase P-G and RNase P-U proteins, and RNase P RNA were separately treated with micrococcal nuclease to determine if the protein preparation contained any contaminating RNA component of E. coli. After the treatment, micrococcal nuclease was inactivated by addition of EGTA to the reaction as it requires calcium ions for activity. The components were separately treated with proteinase K also to confirm that the activity seen with RNase P-G protein was within the protein. After treatment with nuclease and proteinase, the treated components were used to assay the pre-tRNA processing activity using standard conditions. The activity of RNase P-G protein alone was abolished upon treatment with proteinase K, however it was unaffected after treatment with micrococcal nuclease (Fig 5). As seen before, RNase P-U protein did not show any activity by itself under any conditions (Fig 5). The RNase P RNA alone activity was abolished upon treatment with micrococcal nuclease, and remained unaffected after treatment with proteinase K (Fig 5). When the holoenzymes, reconstituted with RNA component and RNase P-G and PU proteins were treated with proteinase K, their activities were lost (Fig 5). However, treatment of holoenzymes with nuclease resulted in the loss activity of that with P-U protein, whereas there was activity in RNase P-G holoenzyme confirming it to be the activity of RNase P-G protein alone (Fig 5).

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