Metal-phosphate interactions in the hammerhead ribozyme observed by 31P NMR and phosphorothioate substitutions.
Metal-phosphate interactions in the hammerhead ribozyme observed by 31P NMR and phosphorothioate substitutions.
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DOI：
10.1021/bi001249w
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发表时间：
2000-09
期刊：
影响因子：
2.9
通讯作者：
M. Maderia;Laura M. Hunsicker;V. DeRose
中科院分区：
文献类型：
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作者：
M. Maderia;Laura M. Hunsicker;V. DeRose
锤头状核酶是一种催化RNA，在中等离子强度下需要二价金属阳离子才能发挥活性。锤头状核酶中与金属离子结合的两个重要位点是位于茎II和保守核心交界处的A9/G10.1位点和剪刀型磷酸盐(P1.1)。(31)结合硫代取代的P-核磁共振波谱对这些可能的金属中心进行了研究。硫代磷的(31)P核磁共振特征出现在一个独特的光谱窗口中，可以监测添加金属时的变化。通过A9-S(RP)或A9-S(S)(RP)取代将1-2当量的Cd(2+)加到锤头上，导致(31)P核磁共振的前场位移2-3ppm。相反，P1.1-S(Rp)和P1.1-S(Sp)(31)的P核磁共振特征略有移动，方向相反，总的增量变化为~lt；/=0.6ppm，添加高达10当量的Cd(2+)。当添加Cd(2+)时，观察到具有单一的内部硫代修饰的RNA.RNA双链没有显著的位移。用模型化合物包括磷酸二乙酯/硫代磷酸酯、AMP和AMPS得到的数据表明，Cd(2+)-S相互作用产生了(31)P核磁共振的前场位移，即使在弱配位的情况下，如与硫代磷酸二乙酯。综上所述，这些数据预测Cd(2+)对A9位点具有高亲和力，并表明结合口袋中的金属配位具有灵活性。Cd(2+)与裂解位点P1.1-S的相互作用较弱，表现为立体特异性。这些数据对已经提出的解释金属离子对锤头状核酶活性影响的机制有一定的意义。这些实验还表明，(31)P-核磁共振波谱与硫代磷作为核酸中金属结合部位的探针具有潜在的实用价值。
The hammerhead ribozyme is a catalytic RNA that requires divalent metal cations for activity under moderate ionic strength. Two important sites that are proposed to bind metal ions in the hammerhead ribozyme are the A9/G10.1 site, located at the junction between stem II and the conserved core, and the scissile phosphate (P1.1). (31)P NMR spectroscopy in conjunction with phosphorothioate substitutions is used in this study to investigate these putative metal sites. The (31)P NMR feature of a phosphorothioate appears in a unique spectral window and can be monitored for changes upon addition of metals. Addition of 1-2 equiv of Cd(2+) to the hammerhead with an A9-S(Rp) or A9-S(S)(Rp) substitution results in a 2-3 ppm upfield shift of the (31)P NMR resonance. In contrast, the P1.1-S(Rp) and P1.1-S(Sp) (31)P NMR features shift slightly and in opposite directions, with a total change in delta of </=0.6 ppm with addition of up to 10 equiv of Cd(2+). No significant shifts are observed for an RNA.RNA duplex with a single, internal phosphorothioate modification upon addition of Cd(2+). Data obtained using model compounds including diethyl phosphate/thiophosphate, AMP, and AMPS, show that a Cd(2+)-S interaction yields an upfield shift for the (31)P NMR resonance, even in the case of a weak coordination such as with diethyl thiophosphate. Taken together, these data predict that Cd(2+) has a high affinity for the A9 site and suggest that there is flexibility in metal coordination within the binding pocket. Cd(2+) interactions with the cleavage site P1.1-S positions are weaker and appear to be stereospecific. These data have implications for mechanisms that have been proposed to explain the influence of metal ions on hammerhead ribozyme activity. These experiments also show the potential utility of (31)P NMR spectroscopy in conjunction with phosphorothioates as a probe for metal binding sites in nucleic acids.