NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis.
NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis.
复制标题
RNase P RNA 的 NAIM 和位点特异性官能团修饰分析：保守的 P1-P4 多螺旋连接内的镁依赖性结构有助于催化。
DOI：
10.1021/bi012158h
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Harris,MichaelE
中科院分区：
文献类型：
--
作者：
Kaye,NicholasM;Christian,EricL;Harris,MichaelE
tRNA加工核酸内切酶核糖核酸酶P含有一个必需的高度保守的RNA分子（RNase P RNA），它是该酶的催化亚基。为了鉴定和表征参与RNase P RNA催化的功能基团，我们在大肠杆菌RNase P RNA的基础上，在核苷酸类似物干扰作图（NAIM）和位点特异性修饰实验中应用自切割核酶-底物缀合物。在高单价离子浓度（3 M），促进蛋白质独立的底物结合，我们发现，核酶在很大程度上是不敏感的类似物取代和Mg 2+的浓度（1.25 mM）远低于最佳催化速率（>100 mM）所需的产生干扰效应，因为修改的核苷酸碱基。在1.25 mM Mg 2+的反应速率的pH依赖性的检查表明，类似物干扰的敏感性增加是不是由于在限速步骤的变化。在这些条件下，NAIM检测到的核苷酸位置仅位于催化结构域，与核酶的整体结构一致，并且主要发生在高度保守的P1−P4多螺旋连接中。J3/4和J2/4中的几个敏感位置靠近先前在P1-P4元件中确定的二价金属离子结合位点。在J3/4中具有位点特异性N7-deazaadenosine和deazaguanosine修饰的核酶的动力学分析一般与干扰结果一致，并且还允许分析NAIM无法访问的位点。这些结果表明，仅在该区域中，A62、A65和A66的N7位置的修饰导致对反应速率的可测量的影响，并且在每个位置处的修饰对Mg 2+浓度显示出不同的敏感性。这些结果揭示了催化结构域中对底物裂解特别重要的单个官能团的有限子集，并证明了高度保守的P1−P4多螺旋结中催化功能和金属离子依赖性结构之间的密切联系。
The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme−substrate conjugates, on the basis of the RNase P RNA fromEscherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+(1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1−P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1−P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1−P4 multihelix junction.