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首页> 外文期刊>Biochemistry >Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity
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Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity

机译：电子顺磁共振波谱法测定锤头核酶与Mn2 +的结合亲和力及其与裂解活性的关系

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Efficient phosphodiester bond cleavage activity by the hammerhead ribozyme requires divalent cations. Toward understanding this metal ion requirement, the Mn2+-binding properties of hammerhead model ribozymes have been investigated under dilute solution conditions, using electron paramagnetic resonance spectroscopy (EPR) to detect free Mn2+ in the presence of added ribozyme. Numbers and affinities of bound Mn2+ were obtained at pH 7.8 (5 mc triethanolamine) in the presence of 0, 0.1, and 1.0 M NaCl for an RNA-DNA model consisting of a 13-nucleotide DNA "substrate" hybridized to a 34-nucleotide RNA "enzyme" [Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74]. In 0.1 M NaCl, two classes of Mn2+ sites are found with n(1) = 3.7 +/- 0.4, K-d(1) = 4 +/- 1 mu M (type 1) and n(2) = 5.2 +/- 0.4, k(d(2)) = 460 +/- 130 mu M (type 2). The high-affinity type I sites are confirmed for an active RNA-RNA hybrid (34-nucleotide RNA enzyme: 13-nucleotide RNA substrate) by EPR measurements at low Mn2+ concentrations. Decreasing NaCl concentration results in an increased number of bound Mn2+ per hammerhead. By contrast, a binding titration in 1 M NaCl indicates that a single Mn2+ site with apparent K-d similar to 10 mu M is populated in low concentrations of Mn2+, and apparent cooperative effects at higher Mn2+ concentrations result in population of a similar total number of Mn2+ sites (n(t) = 8-10) as found in 0.1 M NaCl. Mn2+-dependent activity profiles are similar for the active RNA-RNA hybrid in 0.1 and 1 M NaCl. Correlation with binding affinities determined by EPR indicates that hammerhead activity in 0.1 M NaCl is only observed after all four of the high-affinity Mn2+ sites are occupied, rises with population of the type 2 sites, and is independent of Mn2+ concentrations corresponding to >8-9 Mn2+ bound per hammerhead. Equivalent measurements in 1 M NaCl demonstrate a rise in activity with the cooperative transition observed in the Mn2+ binding curve. These measurements indicate that, over this NaCl concentration range, hammerhead ribozyme activity is influenced by population of a specific set of divalent cation sites. [References: 51]
机译：锤头状核酶有效的磷酸二酯键裂解活性需要二价阳离子。为了理解这种金属离子的需求，已经在稀溶液条件下研究了锤头型核酶的Mn2 +结合特性，使用电子顺磁共振波谱（EPR）在添加的核酶存在下检测游离的Mn2 +。对于pH 7.8（5 mc三乙醇胺），在0、0.1和1.0 M NaCl的存在下，对于由13个核苷酸的DNA“底物”与34个核苷酸杂交的RNA-DNA模型，可以获得结合的Mn2 +的数量和亲和力RNA“酶” [Pley，HW，Flaherty，KM，和McKay，DB（1994）Nature 372，68-74]。在0.1 M NaCl中，发现两类Mn2 +位点，其中n（1）= 3.7 +/- 0.4，Kd（1）= 4 +/- 1μM（类型1）和n（2）= 5.2 +/- 0.4，k（d（2））＝ 460 +/-130μM（类型2）。通过在低Mn2 +浓度下进行EPR测量，确认了活性RNA-RNA杂合体（34个核苷酸的RNA酶：13个核苷酸的RNA底物）的高亲和力I型位点。降低NaCl浓度会导致每个锤头结合Mn2 +的数量增加。相比之下，在1 M NaCl中的结合滴定表明，在低浓度的Mn2 +中存在一个表观Kd类似于10μM的Mn2 +单个位点，而在较高的Mn2 +浓度下明显的协同作用导致总Mn2 +总数相似位点（n（t）= 8-10），位于0.1 M NaCl中。在0.1和1 M NaCl中，活性RNA-RNA杂合体的Mn2 +依赖性活性谱相似。与通过EPR确定的结合亲和力的相关性表明，只有在所有四个高亲和力Mn2 +位点都被占据后，才能在0.1 M NaCl中观察到锤头活性，随着2型位点的增加而升高，并且独立于Mn2 +浓度，对应于> 8每个锤头结合-9 Mn2 +。在1 M NaCl中进行的等效测量表明，随着Mn2 +结合曲线中观察到的协同跃迁，活性升高。这些测量结果表明，在此NaCl浓度范围内，锤头状核酶的活性受一组特定的二价阳离子位点的影响。 [参考：51]

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