TAL effector specificity for base 0 of the DNA target is altered in a complex, effector- and assay-dependent manner by substitutions for the tryptophan in cryptic repeat -1.
TAL effector specificity for base 0 of the DNA target is altered in a complex, effector- and assay-dependent manner by substitutions for the tryptophan in cryptic repeat -1.
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DOI：
10.1371/journal.pone.0082120
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Bogdanove AJ
中科院分区：
文献类型：
--
作者：
Doyle EL;Hummel AW;Demorest ZL;Starker CG;Voytas DF;Bradley P;Bogdanove AJ
TAL效应子是用于定制基因调控的可重靶向转录因子，并且作为TAL效应子-核酸酶融合体（TALEN）用于基因组工程。它们的标志性特征是一个可定制的多态性氨基酸重复序列的中心串，这些重复序列与单个DNA碱基一对一地相互作用以指定目标。在自然界中，TAL效应子重复序列靶向的序列几乎都直接在最大活性所需的胸腺嘧啶（T）之前，并且通常在这种约束下选择定制TAL效应子构建体的靶位点。多个晶体结构表明，在碱基0处对T的这种需求是由中心重复序列N-末端的隐蔽重复序列中的色氨酸残基（W232）编码的，该中心重复序列与T表现出能量上有利的货车范德华接触。我们基于TAL效应子PthXo 1产生了W232的所有单个氨基酸取代的变体。在转录激活试验中，许多取代改变或放松了对T的特异性，少数取代与野生型一样活跃。有些人表现出更高的活动。然而，当在不同的TAL效应子中复制时，取代的效果不同。此外，当在DNA切割测定中的TALEN背景下和在TAL效应物-DNA结合测定中测试时，效果不同。用青枯雷尔氏菌的TAL效应子样蛋白之一的N-末端区域取代PthXo 1构建体的N-末端区域，其具有精氨酸代替色氨酸，导致对作为5'碱基的鸟嘌呤的特异性，但活性低，并且对精氨酸的几个取代，包括色氨酸，完全破坏活性。因此，在W232（或等同）位置的取代对特异性和活性产生的影响是复杂的，并依赖于上下文。产生具有高活性的TAL效应物支架，其稳健地适应在位置0处没有T的位点，可能需要更大规模的再工程。
TAL effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL effector constructs have typically been selected with this constraint. Multiple crystal structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL effector-like proteins of Ralstonia solanacearum, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5’ base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.
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影响因子：
3.7
作者：
Geissler R;Scholze H;Hahn S;Streubel J;Bonas U;Behrens SE;Boch J
通讯作者：
Boch J
Tale-DNA相互作用的定量分析表明极性效应。
影响因子：
14.9
作者：
Meckler JF;Bhakta MS;Kim MS;Ovadia R;Habrian CH;Zykovich A;Yu A;Lockwood SH;Morbitzer R;Elsäesser J;Lahaye T;Segal DJ;Baldwin EP
通讯作者：
Baldwin EP
影响因子：
14.9
作者：
Li T;Huang S;Jiang WZ;Wright D;Spalding MH;Weeks DP;Yang B
通讯作者：
Yang B
影响因子：
3.7
作者：
Blount BA;Weenink T;Vasylechko S;Ellis T
通讯作者：
Ellis T
DOI：
10.1073/pnas.1019533108
发表时间：
2011-02-08
影响因子：
11.1
作者：
Mahfouz, Magdy M.;Li, Lixin;Zhu, Jian-Kang
通讯作者：
Zhu, Jian-Kang