The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers.
The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers.
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DOI：
10.1093/nar/gkw421
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发表时间：
2016-09-06
影响因子：
14.9
通讯作者：
Beisel CL
中科院分区：
文献类型：
--
作者：
Luo ML;Jackson RN;Denny SR;Tokmina-Lukaszewska M;Maksimchuk KR;Lin W;Bothner B;Wiedenheft B;Beisel CL
细菌和古菌通过将外来DNA片段整合到CRISPR(成簇的规则间隔的短回文重复)基因座中来获得对外来遗传元素的抵抗力。在大肠杆菌中，CRISPR衍生的RNAs(CrRNAs)与Cas蛋白组装成一个多亚单位监测复合体，称为Cascade(CRISPR相关抗病毒防御复合体)。CASCADE通过蛋白质介导的Protspacer邻近基序的识别以及crRNA间隔区和DNA靶标之间的互补碱基配对来识别DNA靶标。先前确定的Cascade结构表明，crRNA沿着寡聚蛋白质组装而拉伸，这让我们想知道crRNA的长度如何影响这个复合体的组装和功能。我们发现，延长crRNA的间隔区部分会导致更大的级联复合体，其化学计量比发生了变化，并在体外保持了与目标DNA的结合亲和力。更长的间隔区也保留了Cascade在体内抑制靶基因表达和招募Cas3核酸内切酶降解靶基因的能力。最后，较长的间隔区在特定的靶位置表现出更强的沉默，并且对扩展区域内的失配敏感。这些发现证明了I-E型CRISPR机械的灵活性，并建议可以修改间隔器长度来微调级联活动。
Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.
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影响因子：
3.5
作者：
CHEREPANOV, PP;WACKERNAGEL, W
通讯作者：
WACKERNAGEL, W
影响因子：
16
作者：
Leenay RT;Maksimchuk KR;Slotkowski RA;Agrawal RN;Gomaa AA;Briner AE;Barrangou R;Beisel CL
通讯作者：
Beisel CL
影响因子：
3.8
作者：
Luo ML;Leenay RT;Beisel CL
通讯作者：
Beisel CL
影响因子：
16
作者：
Blosser, Timothy R.;Loeff, Luuk;Westra, Edze R.;Vlot, Marnix;Kunne, Tim;Sobota, Malgorzata;Dekker, Cees;Brouns, Stan J. J.;Joo, Chirlmin
通讯作者：
Joo, Chirlmin
影响因子：
4.5
作者：
Hale, Caryn;Kleppe, Kyle;Terns, Michael P.
通讯作者：
Terns, Michael P.