Sherrier Timothy W, Barth Valdir C, Schifano Jason M, Greendyk Julia R, Woychik Nancy A
Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
J Biol Chem. 2025 Jul;301(7):110323. doi: 10.1016/j.jbc.2025.110323. Epub 2025 May 30.
The genome of the bacterial pathogen that causes tuberculosis, Mycobacterium tuberculosis (Mtb), encodes an inexplicably high number of type II toxin-antitoxin (TA) systems. Because this ancient pathogen has evolved to resist clearance by antibiotics and the host immune system, its TA systems are thought to participate in the survival of these stresses. Of the ∼70 Mtb type II TA systems, 10 MazEF family members have been previously identified, yet the precise cellular target of only one of these MazF toxins is known. Here, we demonstrate that the Rv3098A gene encodes an 11th MazF paralog in Mtb (MazF-mt11, MazF11). As with all MazF toxins, MazF-mt11 acts as a single-strand, sequence-specific endoribonuclease. We first performed primer extension on the large single-stranded MS2 enterobacteriophage RNA substrate after incubation with recombinant MazF-mt11 to identify a single toxin cleavage site between C↓A. We then further pinpointed the boundaries of the MazF-mt11 cleavage consensus sequence as C↓ACCU using Escherichia coli MORE (Mapping by Overexpression of an RNase in E. coli) RNA-seq. Finally, we enlisted 5'-OH RNA-seq to reveal 16S rRNA in the 30S ribosomal subunit as the only MazF-mt11 RNA target in mycobacteria. In fact, the single cleavage site in C↓ACCU maps just before the anti-Shine-Dalgarno sequence at the 3' end of 16S rRNA. Targeted removal of the anti-Shine-Dalgarno sequence by MazF-mt11 leads to nearly complete inhibition of protein synthesis, consistent with its important role in directing ribosomes to translation start codons in leadered mRNAs. The accompanying growth arrest phenotype suggests that MazF-mt11 may participate in establishment of the nonreplicating persistent state in Mtb.
导致结核病的细菌病原体结核分枝杆菌（Mtb）的基因组编码了数量高得令人费解的II型毒素-抗毒素（TA）系统。由于这种古老的病原体已经进化到能够抵抗抗生素和宿主免疫系统的清除，其TA系统被认为参与了应对这些压力时的生存过程。在约70个Mtb II型TA系统中，之前已鉴定出10个MazEF家族成员，但这些MazF毒素中只有一种的精确细胞靶点是已知的。在这里，我们证明Rv3098A基因在Mtb中编码第11个MazF旁系同源物（MazF-mt11，MazF11）。与所有MazF毒素一样，MazF-mt11作为一种单链、序列特异性内切核糖核酸酶发挥作用。我们首先在与重组MazF-mt11孵育后的大型单链MS2噬菌体RNA底物上进行引物延伸，以确定C↓A之间的单个毒素切割位点。然后，我们使用大肠杆菌MORE（通过在大肠杆菌中过表达核糖核酸酶进行定位）RNA测序进一步精确确定了MazF-mt11切割共有序列的边界为C↓ACCU。最后，我们采用5'-OH RNA测序来揭示30S核糖体亚基中的16S rRNA是分枝杆菌中唯一的MazF-mt11 RNA靶点。事实上，C↓ACCU中的单个切割位点正好位于16S rRNA 3'端的反Shine-Dalgarno序列之前。MazF-mt11对反Shine-Dalgarno序列的靶向去除导致蛋白质合成几乎完全受到抑制，这与其在引导核糖体至含前导序列的mRNA中的翻译起始密码子方面的重要作用一致。伴随的生长停滞表型表明MazF-mt11可能参与了Mtb中非复制性持续状态的建立。
