Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis. We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis. Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae. Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc.

中文翻译：

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分枝杆菌中的锌可逆地抑制条件性 DnaB 蛋白剪接。


内含肽作为翻译后调节元件，可以通过条件蛋白剪接（CPS）调节蛋白质功能以适应环境变化。翻译为中断宿主蛋白的子结构域，内含肽剪接以无疤痕地连接侧翼序列（外显肽）。我们使用耻垢分枝杆菌复制解旋酶中的 DnaB-intein1 (DnaBi1) 构建卡那霉素内含肽剪接报告基因 (KISR)，将 DnaBi1 剪接与卡那霉素抗性联系起来。利用异源大肠杆菌中的表达，我们观察到了不同水平的剪接依赖性抗性 (SDR) 的表型类别，并将这些表型类别与卡那霉素抗性蛋白 (KanR) 中 DnaBi1 的插入位置相关联。展示最严格 SDR 的 KanR-DnaBi1 构建体用于探测天然宿主环境耻垢分枝杆菌中 DnaB 的 CPS。我们在此表明​​，锌在分枝杆菌发病机制中很重要，可抑制耻垢分枝杆菌中的 DnaB 剪接。使用体外报告系统，我们证明锌有效且可逆地抑制 DnaBi1 剪接，以及来自麻风分枝杆菌的类似内含肽的剪接。最后，在 1.95 Å 晶体结构中，我们表明锌通过与启动剪接反应的半胱氨酸结合来抑制剪接。总之，我们的结果为分枝杆菌 DnaB 蛋白剪接以及 DNA 复制对环境锌做出反应的模型提供了令人信服的支持。