Department of Biological Sciences and RNA Institute and Department of Biomedical Sciences, School of Public Health , University at Albany , Albany , New York 12222 , United States.
Biochemistry. 2019 Aug 6;58(31):3335-3339. doi: 10.1021/acs.biochem.9b00506. Epub 2019 Jul 23.
Inteins, or intervening proteins, are mobile genetic elements translated within host polypeptides and removed through protein splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional protein splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading protein function. The RadA recombinase from the archaeon represents an intriguing example of CPS, whereby protein splicing is inhibited by interactions between the intein and host protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the splicing rate and accuracy. Here, we investigate the mechanism by which ssDNA exhibits this influence and find that ssDNA strongly promotes a specific step of the splicing reaction, cyclization of the terminal asparagine of the intein. Interestingly, inhibitory interactions between the host protein and intein that block splicing localize to this asparagine, suggesting that ssDNA binding alleviates this inhibition to promote splicing. We also find that ssDNA directly influences the position of catalytic nucleophiles required for protein splicing, implying that ssDNA promotes assembly of the intein active site. This work advances our understanding of how ssDNA accelerates RadA splicing, providing important insights into this intriguing example of CPS.
内含子，或介入蛋白，是可移动的遗传元件，在宿主多肽内翻译，并通过蛋白剪接去除。这个自我催化的过程会打破两个肽键，并将侧翼序列（称为 N-和 C-外显子）重新连接起来，内含子会毫不费力地从宿主蛋白中逃脱。由于这些元件传统上被视为纯粹自私的遗传元件，最近的研究表明，几种天然存在的内含子的条件性蛋白剪接（CPS）可以被与宿主生物体生存相关的各种环境线索或入侵蛋白功能至关重要的线索所调节。古细菌中的 RadA 重组酶就是 CPS 的一个有趣例子，其中蛋白剪接被内含子和宿主蛋白 C-外显子之间的相互作用所抑制。单链 DNA（ssDNA）是 RadA 的天然底物，也是细胞需要重组酶活性的信号，它极大地提高了剪接的速度和准确性。在这里，我们研究了 ssDNA 表现出这种影响的机制，发现 ssDNA 强烈促进了剪接反应的一个特定步骤，即内含子末端天冬酰胺的环化。有趣的是，抑制剪接的宿主蛋白和内含子之间的相互作用定位于这个天冬酰胺，这表明 ssDNA 结合可以缓解这种抑制作用，从而促进剪接。我们还发现 ssDNA 直接影响蛋白剪接所需的催化亲核试剂的位置，这意味着 ssDNA 促进了内含子活性位点的组装。这项工作增进了我们对 ssDNA 如何加速 RadA 剪接的理解，为这一有趣的 CPS 提供了重要的见解。
