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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

CD spectroscopy of RNase P protein components.A. Far-UV CD spectra of the two protein preparations from 205–250 nm at different temperatures. B. CD profile of RNase P-U protein and RNase P-G protein within the temperature range of 20–80°C. C. Near-UV CD spectra of RNase P RNA (2 μM), and RNase P-U protein and RNase P-G protein (12 μM each) within 250–320 nm.
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pone.0153798.g007: CD spectroscopy of RNase P protein components.A. Far-UV CD spectra of the two protein preparations from 205–250 nm at different temperatures. B. CD profile of RNase P-U protein and RNase P-G protein within the temperature range of 20–80°C. C. Near-UV CD spectra of RNase P RNA (2 μM), and RNase P-U protein and RNase P-G protein (12 μM each) within 250–320 nm.

Mentions: CD spectroscopy of the two protein preparations was done at 25°C and 65°C (Fig 7A). Both the proteins showed similar structure at 25°C (Fig 7A). However, when exposed to 65°C, RNase P-G protein lost considerable secondary structure compared to that at 25°C, whereas RNase P-U protein did not lose its secondary structure significantly at 65°C (Fig 7A). At 65°C, the CD spectra for both the proteins showed noise beyond 205 nm, so scans have been shown for the 205–250 nm region.


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)

CD spectroscopy of RNase P protein components.A. Far-UV CD spectra of the two protein preparations from 205–250 nm at different temperatures. B. CD profile of RNase P-U protein and RNase P-G protein within the temperature range of 20–80°C. C. Near-UV CD spectra of RNase P RNA (2 μM), and RNase P-U protein and RNase P-G protein (12 μM each) within 250–320 nm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153798.g007: CD spectroscopy of RNase P protein components.A. Far-UV CD spectra of the two protein preparations from 205–250 nm at different temperatures. B. CD profile of RNase P-U protein and RNase P-G protein within the temperature range of 20–80°C. C. Near-UV CD spectra of RNase P RNA (2 μM), and RNase P-U protein and RNase P-G protein (12 μM each) within 250–320 nm.
Mentions: CD spectroscopy of the two protein preparations was done at 25°C and 65°C (Fig 7A). Both the proteins showed similar structure at 25°C (Fig 7A). However, when exposed to 65°C, RNase P-G protein lost considerable secondary structure compared to that at 25°C, whereas RNase P-U protein did not lose its secondary structure significantly at 65°C (Fig 7A). At 65°C, the CD spectra for both the proteins showed noise beyond 205 nm, so scans have been shown for the 205–250 nm region.

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