Institute for Cellular and Molecular Biology and Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA.
Department of Pathology, Stanford University, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.
Mol Cell. 2018 Nov 15;72(4):700-714.e8. doi: 10.1016/j.molcel.2018.09.013. Epub 2018 Oct 18.
Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.
原核生物的 CRISPR-Cas 系统通过将部分外源核酸（间隔序列）整合到基因组 CRISPR 阵列中，从而提供适应性免疫。Cas6 蛋白随后将 CRISPR 阵列转录本加工成间隔序列衍生的 RNA（CRISPR RNA；crRNA），这些 RNA 靶向 Cas 核酸酶以识别匹配的入侵物。我们发现一种 Marinomonas mediterranea 融合蛋白结合了三个酶结构域（Cas6、逆转录酶 [RT] 和 Cas1），这些结构域在 crRNA 生物发生和从 RNA 和 DNA 获得间隔序列方面发挥作用。我们报告了这种不同寻常的 Cas6 的晶体结构，确定了 Cas6 活性所必需的氨基酸，表明 Cas6 结构域对于 RT 活性和 RNA 间隔序列的获取是必需的，并证明了 CRISPR 重复序列与 Cas6 的结合调节 RT 活性。假定相互作用表面的共同进化表明 Cas6 和 RT 结构域之间存在特定的结构相互作用，系统发育分析揭示了游离 Cas6 与 CRISPR RT 在多个微生物谱系中的重复、稳定关联，表明在融合进化之前，这些蛋白之间存在功能相互作用。
