ROLE OF DIVALENT METAL-IONS IN THE HAMMERHEAD RNA CLEAVAGE REACTION
ROLE OF DIVALENT METAL-IONS IN THE HAMMERHEAD RNA CLEAVAGE REACTION
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DOI：
10.1021/bi00103a011
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发表时间：
1991-10-01
期刊：
影响因子：
2.9
通讯作者：
UHLENBECK, OC
中科院分区：
文献类型：
--
作者：
DAHM, SC;UHLENBECK, OC
利用由两个寡核苷酸组成的双锤头自裂结构域，研究了二价金属离子在裂解反应中的作用。在不存在精胺或不存在精胺的情况下，以MgCl2、MnCl2和CaCl2浓度的函数来测量切割率。在精胺的存在下，速率与金属离子浓度的曲线更宽，较低浓度的二价离子是催化活性所必需的。这表明精胺可以促进双髻鲨的正常折叠，并且该反应需要一个或多个二价离子。另外测试了六种二价离子支持锤头裂解的能力。在没有精胺的情况下，Co2+的裂解速度很快，而Sr2+和Ba2+的裂解速度很慢。Cd2+、Zn2+或Pb2+未观察到特异性裂解。然而，在0.5 mM精胺的存在下，Zn2+和Cd2+的切割速度很快，而Sr2+的切割速度也有所提高，这表明虽然这三种离子不能促进锤头的正常折叠，但它们能够刺激锤头的切割。这些结果表明，某些二价离子要么直接参与裂解机制，要么专门参与稳定锤头的三级结构。此外，当在裂解位点插入一个独特的硫代磷酸键时，观察到二价金属离子特异性的改变。硫取代非桥接氧原子大大降低了有效裂解所必需的重要Mg2+离子的亲和力。相反，与Mn2+的反应正常进行，可能是由于它能够与氧和硫配合。这表明二价离子直接配位到裂解位点的磷酸基上。
A hammerhead self-cleaving domain composed of two oligoribonucleotides was used to study the role of divalent metal ions in the cleavage reaction. Cleavage rates were measured as a function of MgCl2, MnCl2, and CaCl2 concentration in the absence or presence of spermine. In the presence of spermine, the rate vs metal ion concentration curves are broader, and lower concentrations of divalent ions are necessary for catalytic activity. This suggests that spermine can promote proper folding of the hammerhead and one or more divalent ions are required for the reaction. Six additional divalent ions were tested for their ability to support hammerhead cleavage. In the absence of spermine, rapid cleavage was observed with Co2+ while very slow cleavage occurred with Sr2+ and Ba2+. No detectable specific cleavage was observed with Cd2+, Zn2+, or Pb2+. However, in the presence of 0.5 mM spermine, rapid cleavage was observed with Zn2+ and Cd2+, and the rate with Sr2+ was increased, indicating that while these three ions could not promote proper folding of the hammerhead they were able to stimulate cleavage. These results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead. Additionally, an altered divalent metal ion specificity was observed when a unique phosphorothioate linkage was inserted at the cleavage site. The substitution of a sulfur for a nonbridging oxygen atom substantially reduced the affinity of an important Mg2+ ion necessary for efficient cleavage. In contrast, the reaction proceeds normally with Mn2+, presumably due to its ability to coordinate with both oxygen and sulfur. This suggests that a divalent ion coordinates directly to the phosphate at the cleavage site.