The pre-tRNA nucleotide base and 2'-hydroxyl at N(-1) contribute to fidelity in tRNA processing by RNase P.
The pre-tRNA nucleotide base and 2'-hydroxyl at N(-1) contribute to fidelity in tRNA processing by RNase P.
复制标题
DOI：
10.1016/j.jmb.2004.10.080
复制
发表时间：
2005-02
影响因子：
5.6
通讯作者：
N. Zahler;Lei Sun;E. Christian;M. Harris
中科院分区：
文献类型：
--
作者：
N. Zahler;Lei Sun;E. Christian;M. Harris
核糖核酸酶P处理大肠杆菌tRNA的精确性部分取决于与核碱基和N（−1）的2′羟基的相互作用，N（−1）是RNA链切割位点上游的核苷酸。在这里，我们报告了一系列的生物化学和结构功能的研究，旨在解决这些相互作用如何有助于切割位点的选择。我们发现同时破坏切割位点核碱基和2′羟基相互作用导致平行反应，导致正确切割和错误切割正确位点上游（5′）的一个核苷酸。Mg 2+浓度和pH值的变化可能会影响不正确处理的产品部分，pH值影响可归因于正确和错误裂解反应途径的限速步骤差异。此外，我们提供的证据表明，与邻近反应性磷酸基团的2′羟基的相互作用也有助于在错误切割位点的催化作用。最后，当核酶和N（−1）之间的配对也被破坏时，相邻的2′-羟基接触的破坏对催化作用有更大的影响，同时破坏这些接触对结合的影响也是非加和的。这些结果的一个含义是，错误切割将由活性位点修饰的任何组合引起，所述活性位点修饰使正确切割的速率降低超过一定阈值。事实上，我们发现正确切割和相应的错误切割的抑制也是由于活性位点接触的任何组合的破坏，包括金属离子相互作用和与3′ RCCA序列的保守配对相互作用。维持保真度所需的相互作用中的这种冗余可能反映了体内多底物识别的必要性。这些研究为解释底物修饰对RNase P切割保真度的影响提供了一个框架，并为过渡态中与反应性磷酸基团相邻的核碱基和2′羟基的相互作用提供了证据。
Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2′ hydroxyl group of N(−1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure–function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2′ hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5′) of the correct site. Changes in Mg2+concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2′ hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2′-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(−1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3′ RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2′ hydroxyl group adjacent to the reactive phosphate group in the transition state.