The affinity of magnesium binding sites in the Bacillus subtilis RNase P•pre-tRNA complex is enhanced by the protein subunit
The affinity of magnesium binding sites in the Bacillus subtilis RNase P•pre-tRNA complex is enhanced by the protein subunit
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DOI：
10.1021/bi025553w
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发表时间：
2002-07-30
期刊：
影响因子：
2.9
通讯作者：
Fierke, CA
中科院分区：
文献类型：
--
作者：
Kurz, JC;Fierke, CA
细菌核糖核酸酶 P (RNase P) 的 RNA 亚基需要高浓度的镁离子才能在体外有效催化 tRNA 5'-成熟。体内切割前体 tRNA 所需的 RNase P 蛋白质成分通过直接接触 5'-前导序列来增强前体 tRNA 的结合。通过结合瞬态动力学和平衡结合测量，我们现在证明 RNase P 的蛋白质成分还通过特异性增加与 RNase P.pre-tRNA(Asp) 复合物结合的镁离子的亲和力来促进催化。蛋白质成分不会改变镁离子的数量或表观亲和力，这些镁离子要么与 RNase P RNA 聚阴离子广泛相关，要么是结合成熟 tRNAAsp 所需的。该蛋白质组分也不会改变 RNase P 催化的前 tRNA (AsP) 裂解的 pH 依赖性，这进一步证明该蛋白质组分不会直接稳定催化过渡态。然而，蛋白质亚基确实增加了至少四个镁位点的亲和力，从而稳定了前 tRNA 的结合，并可能稳定了催化作用。此外，这种稳定作用与 RNase P 全酶-前 tRNA 复合物中的 P 蛋白/5'-前导序列接触有关。这些结果表明，蛋白质成分通过结合和定位 pretRNA 间接增强 RNase P-pre-tRNA 复合物的镁亲和力。此外，RNase P 被六氨合钴抑制 (K-1 = 0.11 +/- 0.01 mm)，而镁、锰、钴和锌与六氨合钴竞争以激活 RNase P。这些数据与 RNase P 催化需要至少一种金属-水配体或一种内球金属接触的假设一致。
The RNA subunit of bacterial ribonuclease P (RNase P) requires high concentrations of magnesium ions for efficient catalysis of tRNA 5'-maturation in vitro. The protein component of RNase P, required for cleavage of precursor tRNA in vivo, enhances pre-tRNA binding by directly contacting the 5'-leader sequence. Using a combination of transient kinetics and equilibrium binding measurements, we now demonstrate that the protein component of RNase P also facilitates catalysis by specifically increasing the affinities of magnesium ions bound to the RNase P.pre-tRNA(Asp) complex. The protein component does not alter the number or apparent affinity of magnesium ions that are either diffusely associated with the RNase P RNA polyanion or required for binding mature tRNAAsp. Nor does the protein component alter the pH dependence of pre-tRNA(AsP) cleavage catalyzed by RNase P, providing further evidence that the protein component does not directly stabilize the catalytic transition state. However, the protein subunit does increase the affinities of at least four magnesium sites that stabilize pre-tRNA binding and, possibly, catalysis. Furthermore, this stabilizing effect is coupled to the P protein/5'-leader contact in the RNase P holoenzyme-pre-tRNA complex. These results suggest that the protein component enhances the magnesium affinity of the RNase P-pre-tRNA complex indirectly by binding and positioning pretRNA. Furthermore, RNase P is inhibited by cobalt hexammine (K-1 = 0.11 +/- 0.01 mm) while magnesium, manganese, cobalt, and zinc compete with cobalt hexammine to activate RNase P. These data are consistent with the hypothesis that catalysis by RNase P requires at least one metal-water ligand or one inner-sphere metal contact.