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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

Comparison of pre-tRNA processing by RNase P RNA and RNase P-G protein and zymogram analysis of protein preparations.A. Indicated concentrations of magnesium and ammonium ions were used to assay the pre-tRNA cleavage by RNase P RNA and RNase P-G protein. The reactions contained 50 nM of RNase P RNA and 100 nM of RNase P-G protein. B. Zymogram analysis of the two protein preparations, albumin and RNase A.
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pone.0153798.g004: Comparison of pre-tRNA processing by RNase P RNA and RNase P-G protein and zymogram analysis of protein preparations.A. Indicated concentrations of magnesium and ammonium ions were used to assay the pre-tRNA cleavage by RNase P RNA and RNase P-G protein. The reactions contained 50 nM of RNase P RNA and 100 nM of RNase P-G protein. B. Zymogram analysis of the two protein preparations, albumin and RNase A.

Mentions: One of the reasons for catalytic activity being manifested by RNase P-G protein could be the presence of RNA component of E. coli RNase P which may be co-purifying with the protein. To investigate the possibility of any contaminating RNA in the RNase P-G protein preparation, different combinations of magnesium and ammonium ions were used to assess the pre-tRNA processing by RNase P RNA and RNase P-G protein (Fig 4A). As shown in Fig 4A, RNase P RNA cleaves the pre-tRNA in vitro at high concentrations of magnesium and ammonium ions. On the other hand, RNase P-G protein processed pre-tRNA only at low concentrations of magnesium and ammonium ions, suggesting it to be an activity of the protein (Fig 4A).


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)

Comparison of pre-tRNA processing by RNase P RNA and RNase P-G protein and zymogram analysis of protein preparations.A. Indicated concentrations of magnesium and ammonium ions were used to assay the pre-tRNA cleavage by RNase P RNA and RNase P-G protein. The reactions contained 50 nM of RNase P RNA and 100 nM of RNase P-G protein. B. Zymogram analysis of the two protein preparations, albumin and RNase A.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153798.g004: Comparison of pre-tRNA processing by RNase P RNA and RNase P-G protein and zymogram analysis of protein preparations.A. Indicated concentrations of magnesium and ammonium ions were used to assay the pre-tRNA cleavage by RNase P RNA and RNase P-G protein. The reactions contained 50 nM of RNase P RNA and 100 nM of RNase P-G protein. B. Zymogram analysis of the two protein preparations, albumin and RNase A.
Mentions: One of the reasons for catalytic activity being manifested by RNase P-G protein could be the presence of RNA component of E. coli RNase P which may be co-purifying with the protein. To investigate the possibility of any contaminating RNA in the RNase P-G protein preparation, different combinations of magnesium and ammonium ions were used to assess the pre-tRNA processing by RNase P RNA and RNase P-G protein (Fig 4A). As shown in Fig 4A, RNase P RNA cleaves the pre-tRNA in vitro at high concentrations of magnesium and ammonium ions. On the other hand, RNase P-G protein processed pre-tRNA only at low concentrations of magnesium and ammonium ions, suggesting it to be an activity of the protein (Fig 4A).

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