Kaya Emine, Doxzen Kevin W, Knoll Kilian R, Wilson Ross C, Strutt Steven C, Kranzusch Philip J, Doudna Jennifer A
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;
Biophysics Graduate Group, University of California, Berkeley, CA 94720;
Proc Natl Acad Sci U S A. 2016 Apr 12;113(15):4057-62. doi: 10.1073/pnas.1524385113. Epub 2016 Mar 30.
Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5'-hydroxylated guide RNAs rather than the 5'-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo-RNA complex reveals a guide strand binding site comprising residues that block 5' phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPR-cas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5'-hydroxylated guide.
真核生物的AGO蛋白通过小RNA引导识别和切割mRNA靶标来诱导基因沉默。尽管人类和原核生物AGO蛋白之间的结构相似性与共同的机制特性一致，但基于序列和结构的比对表明，CRISPR - cas（成簇规律间隔短回文重复序列及其相关蛋白）细菌免疫操纵子内编码的AGO蛋白具有不同的活性。我们在此表明，与CRISPR相关的嗜压栖热袍菌AGO（MpAGO）蛋白使用5'-羟基化的引导RNA切割单链靶序列，而不是所有已知AGO蛋白使用的5'-磷酸化引导RNA。MpAGO - RNA复合物的2.0埃分辨率晶体结构揭示了一个引导链结合位点，该位点包含阻止5'磷酸相互作用的残基。通过基于结构的序列比对，我们能够鉴定出其他推定的类似MpAGO的蛋白，所有这些蛋白都在CRISPR - cas基因座内编码。综上所述，我们的数据表明存在一类对5'-羟基化引导具有非规范特异性的AGO亚类。
