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Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems
Published on: April 26, 2019
Structural, functional and biophysical characterization of two cryptic RNA methyltransferases (Rv3366 and Rv3919c) of
Tasmin Nazim1, Faraz Ahmed1, Md Amjad Beg2
1Department of Molecular Medicine, Jamia Hamdard, New Delhi, India.
Journal of Biomolecular Structure & Dynamics
|July 9, 2026
Summary
Two uncharacterized RNA methyltransferases (MTases) from Mycobacterium tuberculosis, Rv3366 and Rv3919c, were found to be functional enzymes. They exhibit methyltransferase activity and RNA binding, suggesting roles in bacterial virulence and adaptation.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- RNA methyltransferases (MTases) are crucial for bacterial post-transcriptional regulation, impacting virulence and adaptation.
- Several putative MTases in Mycobacterium tuberculosis lack functional characterization, hindering understanding of its physiology and pathogenicity.
Purpose of the Study:
- To characterize the structural, biophysical, and functional properties of two predicted M. tuberculosis RNA MTases, Rv3366 and Rv3919c.
- To elucidate their potential roles in mycobacterial physiology and pathogenicity.
Main Methods:
- Cloning and heterologous expression of Rv3366 and Rv3919c genes in Escherichia coli.
- Purification using affinity chromatography.
- Enzymatic activity assays (Methyltransferase-Glo™) with S-adenosylmethionine (SAM).
- Biophysical and structural characterization using circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and thermal shift assays.
Main Results:
- Both Rv3366 and Rv3919c demonstrated SAM-dependent methyltransferase activity.
- Proteins showed significant RNA-binding affinity.
- CD spectroscopy indicated a predominantly α-helical secondary structure.
- Fluorescence and thermal shift assays confirmed proper folding and thermal stability.
Conclusions:
- Rv3366 and Rv3919c possess characteristics of functional RNA MTases.
- These enzymes likely participate in RNA modification pathways contributing to M. tuberculosis virulence and adaptation.
- Findings provide a basis for further mechanistic studies and potential therapeutic target evaluation.
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