Department of Biochemistry and Cell Biology, Robert Johnson Medical School, Rutgers University, Piscataway, NJ, 08854, USA; Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ, 08854, USA.
Department of Computer Science, University of Massachusetts, Boston, MA, 02125, USA.
Biochem Biophys Res Commun. 2019 Oct 20;518(3):533-540. doi: 10.1016/j.bbrc.2019.08.076. Epub 2019 Aug 21.
MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease.
MazF 是一种序列特异性内切核酸酶或 mRNA 干扰酶，可在特定序列处切割 RNA。由于特定基因或特定基因群的表达可以受到 MazF 的调控，因此，对于生物技术和医学应用来说，扩大 MazF mRNA 干扰酶的识别序列库是非常可取的。在这里，我们从极端嗜盐古菌 Methanohalobium evestigatum 中鉴定出一种 MazF 同源物（MazFme）的基因。为了抑制 MazFme 对大肠杆菌细胞的毒性，将同源解毒蛋白 MazEme 的 C 端半部分融合到 MazFme 的 N 端。由于将 MazEme 的 C 端半部分融合到 MazFme 上能够中和 MazFme 的毒性，因此能够大量表达无毒性的 MazEme-MazFme 融合蛋白。纯化 MazEme 后，通过引物延伸和合成核糖核苷酸确定了游离 MazFme 的 RNA 切割特异性，揭示了 MazFme 是一种 CUGGU/UUGGU 特异性内切核酸酶。
