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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

Effect of temperature on the activity of the protein preparations.The two protein preparations, 400 nM each were pre-incubated at indicated temperatures and further used to reconstitute holoenzymes with the 200 nM RNA component. A. RNase P-U holoenzyme. B. RNase P-G holoenzyme. The % product formation in each case was normalized with respect to that at 37°C, which is shown as 100%. Data are plotted as Mean ± SD of three independent observations.
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pone.0153798.g006: Effect of temperature on the activity of the protein preparations.The two protein preparations, 400 nM each were pre-incubated at indicated temperatures and further used to reconstitute holoenzymes with the 200 nM RNA component. A. RNase P-U holoenzyme. B. RNase P-G holoenzyme. The % product formation in each case was normalized with respect to that at 37°C, which is shown as 100%. Data are plotted as Mean ± SD of three independent observations.

Mentions: In order to assess the stability of the two RNase P proteins, they were pre-incubated at 37°, 45°, 50° and 65°C for 30 minutes and subsequently, reconstituted with RNase P RNA to form holoenzymes. While the RNase P-U holoenzyme retained similar pre-tRNA processing activity even after the protein being exposed to high temperatures (Fig 6A), RNase P-G holoenzyme lost considerable activity at 65°C compared to that at 37°C (Fig 6B). This shows that the two protein preparations differ with respect to their stability at higher temperatures.


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)

Effect of temperature on the activity of the protein preparations.The two protein preparations, 400 nM each were pre-incubated at indicated temperatures and further used to reconstitute holoenzymes with the 200 nM RNA component. A. RNase P-U holoenzyme. B. RNase P-G holoenzyme. The % product formation in each case was normalized with respect to that at 37°C, which is shown as 100%. Data are plotted as Mean ± SD of three independent observations.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153798.g006: Effect of temperature on the activity of the protein preparations.The two protein preparations, 400 nM each were pre-incubated at indicated temperatures and further used to reconstitute holoenzymes with the 200 nM RNA component. A. RNase P-U holoenzyme. B. RNase P-G holoenzyme. The % product formation in each case was normalized with respect to that at 37°C, which is shown as 100%. Data are plotted as Mean ± SD of three independent observations.
Mentions: In order to assess the stability of the two RNase P proteins, they were pre-incubated at 37°, 45°, 50° and 65°C for 30 minutes and subsequently, reconstituted with RNase P RNA to form holoenzymes. While the RNase P-U holoenzyme retained similar pre-tRNA processing activity even after the protein being exposed to high temperatures (Fig 6A), RNase P-G holoenzyme lost considerable activity at 65°C compared to that at 37°C (Fig 6B). This shows that the two protein preparations differ with respect to their stability at higher temperatures.

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