Growth and Translation Inhibition through Sequence-specific RNA Binding by Mycobacterium tuberculosis VapC Toxin

• Published in: The Journal of biological chemistry Vol. 287; no. 16; pp. 12835 - 12847
• Main Authors: Sharp, Jared D., Cruz, Jonathan W., Raman, Sahadevan, Inouye, Masayori, Husson, Robert N., Woychik, Nancy A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.04.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Antitoxin; Antitoxins - genetics; Antitoxins - metabolism; Bacterial Toxins; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; DNA, Bacterial - metabolism; Enzymology; Escherichia coli - genetics; Latency; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - growth & development; Mycobacterium tuberculosis - metabolism; Protein Biosynthesis - physiology; Protein Synthesis; Ribonuclease; Ribosomes - genetics; Ribosomes - metabolism; RNA; RNA, Bacterial - metabolism; RNA, Messenger - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Translation; Tuberculosis; Virulence Factors; Virulence Factors - genetics; Virulence Factors - metabolism; Translation; Protein Synthesis; Tuberculosis; RNA; Bacterial Toxins; Antitoxin; Ribonuclease; Virulence Factors; Latency
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.340109

The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA. Background:Mycobacterium tuberculosis harbors a highly expanded number of toxin-antitoxin (TA) systems. Results: The M. tuberculosis VapC-mt4 toxin blocks translation and arrests growth through RNA binding at a short recognition sequence. Conclusion:M. tuberculosis VapC toxins have a function distinct from other characterized TA toxins. Significance: TA systems may contribute to the slow growth and dormancy characteristic of M. tuberculosis during latent tuberculosis.