Limits...
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 RNase P-G and RNase P-U holoenzyme complexes at different ammonium acetate concentrations.The pre-tRNA processing activity of RNase P-G and RNase P-U proteins and their holoenzymes was assayed in the presence of different concentrations of ammonium acetate. To reconstitute the RNase P holoenzyme, 50 nM RNase P RNA and 100 nM RNase P protein were used. For protein alone activity, 100 nM protein was used in the assays.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835064&req=5

pone.0153798.g003: Activity of RNase P-G and RNase P-U holoenzyme complexes at different ammonium acetate concentrations.The pre-tRNA processing activity of RNase P-G and RNase P-U proteins and their holoenzymes was assayed in the presence of different concentrations of ammonium acetate. To reconstitute the RNase P holoenzyme, 50 nM RNase P RNA and 100 nM RNase P protein were used. For protein alone activity, 100 nM protein was used in the assays.

Mentions: The activity of the holoenzyme complexes reconstituted separately with RNA component and RNase P-U and RNase P-G protein components was checked on pre-tRNA substrate at various concentrations of ammonium acetate (Fig 3). Both holoenzymes showed pre-tRNA processing activity with increasing ammonium acetate concentration (Table 1). The activity of RNase P-G protein alone was inhibited with increasing concentration of ammonium acetate and it had negligible activity at 500 mM ammonium acetate (Fig 3, Table 1). The RNase P-U protein by itself did not show any pre-tRNA cleavage at any ammonium acetate concentration (Fig 3).


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 RNase P-G and RNase P-U holoenzyme complexes at different ammonium acetate concentrations.The pre-tRNA processing activity of RNase P-G and RNase P-U proteins and their holoenzymes was assayed in the presence of different concentrations of ammonium acetate. To reconstitute the RNase P holoenzyme, 50 nM RNase P RNA and 100 nM RNase P protein were used. For protein alone activity, 100 nM protein was used in the assays.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153798.g003: Activity of RNase P-G and RNase P-U holoenzyme complexes at different ammonium acetate concentrations.The pre-tRNA processing activity of RNase P-G and RNase P-U proteins and their holoenzymes was assayed in the presence of different concentrations of ammonium acetate. To reconstitute the RNase P holoenzyme, 50 nM RNase P RNA and 100 nM RNase P protein were used. For protein alone activity, 100 nM protein was used in the assays.
Mentions: The activity of the holoenzyme complexes reconstituted separately with RNA component and RNase P-U and RNase P-G protein components was checked on pre-tRNA substrate at various concentrations of ammonium acetate (Fig 3). Both holoenzymes showed pre-tRNA processing activity with increasing ammonium acetate concentration (Table 1). The activity of RNase P-G protein alone was inhibited with increasing concentration of ammonium acetate and it had negligible activity at 500 mM ammonium acetate (Fig 3, Table 1). The RNase P-U protein by itself did not show any pre-tRNA cleavage at any ammonium acetate concentration (Fig 3).

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