Interaction of the vaccinia virus nucleoside triphosphate phosphohydrolase I with linear oligonucleotides.
Interaction of the vaccinia virus nucleoside triphosphate phosphohydrolase I with linear oligonucleotides.
复制标题
痘苗病毒核苷三磷酸磷酸水解酶 I 与线性寡核苷酸的相互作用。
DOI：
10.1021/bi9903749
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Niles,EG
中科院分区：
文献类型：
--
作者：
Christen,LA;Sanders,M;Niles,EG
牛痘病毒核苷三磷酸磷酸水解酶I（NPH I）用作早期基因转录终止中使用的ATP酶活性[Deng，L.，和Shuman，S.（1998）Genes Dev.12，538 - 546; Christen，L. M.，等人（1998）Virology 245，360 - 371]。由于ATP酶的活性需要单链DNA的结合，充分了解寡核苷酸激活的机制是阐明其在转录终止中的作用所必需的。为了启动NPH I的详细结构-功能研究，我们对线性寡核苷酸与NPH I的相互作用进行了动力学和结合分析。在单链DNA的存在下，ATP表现出复杂的饱和动力学。ATP的表观Km与DNA浓度无关，表明ssDNA结合改变了反应的kcat。长度为18至48个核苷酸的线性ssDNA寡核苷酸以可饱和的方式刺激活性。随着寡核苷酸长度的增加，Kact减小，Vmax增加。通过EMSA测量，亲和力的增加被结合水平的增加所抵消。观察到的36个核苷酸的ssDNA的动力学活化依赖于ATP浓度。在低ATP水平下，观察到S形饱和动力学，而在饱和ATP水平下，观察到近双曲线动力学，表明NPH I可能采用两种构象状态。长度为18、24和36个碱基的线性寡核苷酸分别最大限度地结合一个、两个和三个NPH I分子，表明NPH I结合位点的长度不超过12个碱基。相比之下，单链RNA不刺激ATP酶活性，但RNA结合类似长度的DNA。RNA和DNA都可以通过UV光与NPH I光交联。ssDNA和ssRNA在与NPH I的UV光交联中交叉竞争，表明两种寡核苷酸共享共同的结合位点。ssRNA阻止ssDNA激活ATP酶活性，证实两种寡核苷酸均与NPH I上动力学重要的寡核苷酸激活位点结合。ssDNA抑制体外转录终止。通过加入NPH I克服抑制，表明寡核苷酸抑制是通过NPH I介导的。
Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) serves as the ATPase activity employed in early gene transcription termination [Deng, L., and Shuman, S. (1998)Genes Dev.12, 538−546; Christen, L. M., et al. (1998)Virology 245, 360−371]. Since ATPase activity requires binding of single-stranded DNA, a full understanding of the mechanism of oligonucleotide activation is essential for the elucidation of its role in transcription termination. To initiate detailed structure−function studies of NPH I, we undertook combined kinetic and binding analyses of the interaction of linear oligonucleotides with NPH I. In the presence of single-stranded DNA, ATP exhibits complex saturation kinetics. The apparentKmfor ATP is independent of DNA concentration, demonstrating that ssDNA binding alters thekcatfor the reaction. Linear ssDNA oligonucleotides from 18 to 48 nucleotides in length stimulated activity in a saturatable fashion. As the oligonucleotide length increases, theKactdecreases and theVmaxincreases. The increase in affinity is paralleled by an increase in the level of binding as measured by EMSA. The kinetic activation observed for 36-nucleotide ssDNA is dependent upon ATP concentration. At low ATP levels, sigmoidal saturation kinetics are observed, while at saturating ATP levels, near-hyperbolic kinetics are seen, suggesting that NPH I may adopt two conformational states. Linear oligonucleotides 18, 24, and 36 bases in length bind one, two, and three molecules of NPH I maximally, respectively, indicating that the NPH I binding site is no more than 12 bases in length. In contrast, single-stranded RNA does not stimulate ATPase activity, yet RNA binds as well as DNA of a similar length. Both RNA and DNA can be photo-cross-linked to NPH I by UV light. ssDNA and ssRNA cross-compete in UV photo-cross-linking to NPH I, indicating that both oligonucleotides share a common binding site. ssRNA prevents ssDNA activation of ATPase activity, confirming that both oligonucleotides bind to the kinetically important oligonucleotide activation site on NPH I. ssDNA inhibits transcription termination in vitro. Inhibition is overcome by adding NPH I, demonstrating that oligonucleotide inhibition is mediated through NPH I.