Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry, New Jersey (UMDNJ)-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2012 Apr 13;287(16):12835-47. doi: 10.1074/jbc.M112.340109. Epub 2012 Feb 21.
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.
结核分枝杆菌基因组中含有大量的毒素-抗毒素（TA）模块。奇怪的是，其中一半以上是 VapBC（与毒力相关的蛋白）家族成员。尽管如此，VapC 毒素在这种重要病原体中的细胞靶标、精确作用模式和生理作用仍不清楚。为了更好地了解这个毒素家族的功能，我们研究了一个结核分枝杆菌 vapBC-mt4（Rv0596c-Rv0595c）原型 VapBC TA 系统的特征和生化特性。VapC-mt4 的表达导致了大肠杆菌、耻垢分枝杆菌和结核分枝杆菌的生长停滞，这是所有 TA 毒素的一个标志。它的表达导致翻译抑制，伴随着几种 mRNA 的稳态水平逐渐下降。VapC-mt4 在 ACGC 和 AC（A/U）GC 序列的 mRNA 模板上表现出序列特异性内切核酸酶活性。然而，与 TA 毒素 MazF-mt1（Rv2801c）相比，VapC-mt4 的切割活性相对较弱。与其他 TA 毒素不同，翻译抑制和生长停滞先于 mRNA 切割，这表明 VapC-mt4 的 RNA 结合特性而不是 RNA 切割引发了毒性。为了支持这一假设，与通过直接 mRNA 切割抑制翻译的 TA 毒素不同，VapC-mt4 的表达随着时间的推移导致总 RNA 的恢复增加。此外，VapC-mt4 在电泳迁移率变动分析中表现出稳定的、序列特异性的 RNA 结合。最后，VapC-mt4 在无切割相应 mRNA 的情况下在无细胞系统中抑制蛋白质合成。因此，VapC-mt4 的活性在机制上与其他 TA 毒素不同，因为它似乎主要通过选择性、稳定的 RNA 结合来抑制翻译。
