Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity
Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity
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DOI：
10.1021/bi981425p
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发表时间：
1998-12-22
期刊：
影响因子：
2.9
通讯作者：
DeRose, VJ
中科院分区：
文献类型：
--
作者：
Horton, TE;Clardy, DR;DeRose, VJ
锤头核酶的有效磷酸二酯键裂解活性需要二价阳离子。为了了解这种金属离子的要求，我们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) 来检测添加核酶存在下的游离 Mn2+。对于由与 34 核苷酸 RNA“酶”杂交的 13 核苷酸 DNA“底物”组成的 RNA-DNA 模型，在 pH 7.8（5 mc 三乙醇胺）下，在 0、0.1 和 1.0 M NaCl 存在下获得结合的 Mn2+ 的数量和亲和力 [Pley, H. W.、Flaherty, K. M. 和 McKay, D. B. (1994)自然 372, 68-74]。在 0.1 M NaCl 中，发现两类 Mn2+ 位点，n(1) = 3.7 +/- 0.4，K-d(1) = 4 +/- 1 mu M（类型 1）和 n(2) = 5.2 +/- 0.4，k(d(2)) = 460 +/- 130 mu M（类型 2）。通过低 Mn2+ 浓度下的 EPR 测量，确认了活性 RNA-RNA 杂交体（34 核苷酸 RNA 酶：13 核苷酸 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的 Mn2+ 数量增加。相比之下，1 M NaCl 中的结合滴定表明，在低浓度 Mn2+ 中，具有与 10 μM 相似的表观 K-d 的单个 Mn2+ 位点，而较高 Mn2+ 浓度下的明显协同效应导致 Mn2+ 位点总数 (n(t) = 8-10) 与 0.1 M NaCl 中发现的相似。 0.1 和 1 M NaCl 中的活性 RNA-RNA 杂交体的 Mn2+ 依赖性活性特征相似。与 EPR 确定的结合亲和力的相关性表明，仅在所有四个高亲和力 Mn2+ 位点被占据后才观察到 0.1 M NaCl 中的锤头活性，随着 2 型位点的数量增加，并且与对应于每个锤头结合 >8-9 Mn2+ 的 Mn2+ 浓度无关。 1 M NaCl 中的等效测量结果表明，随着 Mn2+ 结合曲线中观察到的协同转变，活性有所增加。这些测量表明，在这个 NaCl 浓度范围内，锤头核酶活性受到一组特定二价阳离子位点的影响。
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.