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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

Activity of protein preparations on pre-tRNA at low ammonium acetate.The pre-tRNA processing activity with different amounts of RNase P protein preparations alone, in the absence of RNA component, was assayed in 50 mM Tris-HCl (pH 7.4), 10 mM magnesium chloride and 100 mM ammonium acetate. S denotes the substrate alone reaction.
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pone.0153798.g002: Activity of protein preparations on pre-tRNA at low ammonium acetate.The pre-tRNA processing activity with different amounts of RNase P protein preparations alone, in the absence of RNA component, was assayed in 50 mM Tris-HCl (pH 7.4), 10 mM magnesium chloride and 100 mM ammonium acetate. S denotes the substrate alone reaction.

Mentions: The two preparations of the RNase P protein component were analyzed for their effect on the pre-tRNA substrate (Fig 2). The RNase P-G protein was found to cleave the pre-tRNA by itself in a dose dependent manner, whereas the RNase P-U protein did not cleave pre-tRNA at all (Fig 2). RNase P-G protein showed complete processing of pre-tRNA beyond 200 nM concentration of the protein (Fig 2).


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)

Activity of protein preparations on pre-tRNA at low ammonium acetate.The pre-tRNA processing activity with different amounts of RNase P protein preparations alone, in the absence of RNA component, was assayed in 50 mM Tris-HCl (pH 7.4), 10 mM magnesium chloride and 100 mM ammonium acetate. S denotes the substrate alone reaction.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153798.g002: Activity of protein preparations on pre-tRNA at low ammonium acetate.The pre-tRNA processing activity with different amounts of RNase P protein preparations alone, in the absence of RNA component, was assayed in 50 mM Tris-HCl (pH 7.4), 10 mM magnesium chloride and 100 mM ammonium acetate. S denotes the substrate alone reaction.
Mentions: The two preparations of the RNase P protein component were analyzed for their effect on the pre-tRNA substrate (Fig 2). The RNase P-G protein was found to cleave the pre-tRNA by itself in a dose dependent manner, whereas the RNase P-U protein did not cleave pre-tRNA at all (Fig 2). RNase P-G protein showed complete processing of pre-tRNA beyond 200 nM concentration of the protein (Fig 2).

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