Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase-helicase from Arabidopsis thaliana.
Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase-helicase from Arabidopsis thaliana.
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由噬菌体 T7 gp4 和来自拟南芥的假定引物酶解旋酶构建的嵌合蛋白。
DOI：
10.1007/s11033-014-3671-y
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发表时间：
2014
影响因子：
2.8
通讯作者：
Crampton,DonaldJ
中科院分区：
文献类型：
--
作者：
Towle-Weicksel,JamieB;Cao,Yun;Crislip,LisaJ;Thurlow,DavidL;Crampton,DonaldJ
拟南芥的一个开放阅读框与人类线粒体DNA解旋酶Flink高度同源，已被克隆、表达，并证明具有DNA启动酶和DNA解旋酶活性。Twinkle Homolog(ATH)的DNA启动酶活性水平较低，可能是由于锌结合结构域(ZBD)不完整所致。在本研究中，ATH的N端截断涉及残基80-102与RNA聚合酶结构域(RPD)相互作用。此外，利用ATH的结构域与特性良好的T7噬菌体DNA启动酶-解旋酶GP4构建了嵌合蛋白，以确定ATH的弱启动子酶活性是否可以增强。构建了两种嵌合蛋白：ATHT7含有ATH的ZBD和RPD结构域与T7的解旋酶结构域相连，而T7ATH含有与ATH的解旋酶结构域相连的T7的ZBD和RPD结构域。成功表达和纯化了两种嵌合蛋白。并对传统的底物酶和解旋酶活性进行了测定。T7ATH能产生短的寡核苷酸引物，但不能被DNA聚合酶协同延伸。虽然T7ATH含有ATH解旋酶结构域，但它几乎没有功能解旋酶的特征。ATHT7完全缺乏底物酶活性，也只表现出微弱的解旋酶活性。这项工作证明了结构和功能不同结构域之间相互作用的重要性，特别是在重组嵌合蛋白中。
An open reading frame fromArabidopsisthaliana, which is highly homologous to the human mitochondrial DNA helicase TWINKLE, was previously cloned, expressed, and shown to have DNA primase and DNA helicase activity. The level of DNA primase activity of thisArabidopsisTwinkle homolog (ATH) was low, perhaps due to an incomplete zinc binding domain (ZBD). In this study, N-terminal truncations of ATH implicate residues 80–102 interact with the RNA polymerase domain (RPD). In addition, chimeric proteins, constructed using domains from ATH and the well-characterized T7 phage DNA primase–helicase gp4, were created to determine if the weak primase activity of ATH could be enhanced. Two chimeric proteins were constructed: ATHT7 contains the ZBD and RPD domains of ATH tethered to the helicase domain of T7, while T7ATH contains the ZBD and RPD domains of T7 tethered to the helicase domain of ATH. Both chimeric proteins were successfully expressed and purified inE. coli,and assayed for traditional primase and helicase activities. T7ATH was able to generate short oligoribonucleotide primers, but these primers could not be cooperatively extended by a DNA polymerase. Although T7ATH contains the ATH helicase domain, it exhibited few of the characteristics of a functional helicase. ATHT7 lacked primase activity altogether and also demonstrated only weak helicase activities. This work demonstrates the importance of interactions between structurally and functionally distinct domains, especially in recombinant, chimeric proteins.