KINETICS AND THERMODYNAMICS OF THE RNASE-P RNA CLEAVAGE REACTION - ANALYSIS OF TRANSFER-RNA 3'-END VARIANTS
KINETICS AND THERMODYNAMICS OF THE RNASE-P RNA CLEAVAGE REACTION - ANALYSIS OF TRANSFER-RNA 3'-END VARIANTS
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DOI：
10.1006/jmbi.1994.0130
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发表时间：
1995-03-24
影响因子：
5.6
通讯作者：
HARTMANN, RK
中科院分区：
文献类型：
--
作者：
HARDT, WD;SCHLEGL, J;HARTMANN, RK
我们研究了（前）tRNA（Gly）的3 '端变体与来自大肠杆菌和嗜热栖热菌的核糖核酸酶P（RNase P）RNA的相互作用。为了从整体催化反应中剖析tRNA结合的热力学，通过凝胶阻滞研究了成熟tRNA（Gly）变体与RNase P RNA的特异性结合。一个新开发的测定，基于减少铅2 +-水解在CCA结束由于复杂的形成tRNA和RNase P RNA，被用来确认的解离常数。结合数据补充了相应的前tRNA（甘氨酸）变体的单次和多次周转动力学分析。对于大肠杆菌RNase P RNA，获得以下结果。通过pCp或三个核苷酸（AUA）的CCA延伸使凝胶解析的tRNA（Gly）结合稳定1至1.5 kcal/mol。将CCA中的第一个C改变为A、G或U导致结合亲和力降低超过100倍，这对应于结合能损失3.5至4.5 kcal/mol。然而，单个周转速率常数仅受到轻微影响，表明tRNA 3 '端介导的与RNase P RNA的相互作用的破坏或损失并不优先使过渡态不稳定。我们的数据表明，另一个动力学步骤后，初始底物结合E。coli RNase P RNA（可能是构象重排）。对于T.在嗜热菌RNase P RNA中，野生型tRNA（Gly）CCAAUA的产物释放在多周转反应中不限速。然而，CCA突变的效果与E. coli RNase P RNA。这支持了这样的观点，即tRNA 3 '端的高亲和力结合位点是真细菌P RNA的普遍特征。最后，这里获得的结果提供了进一步的证据，凝胶阻滞试验是适合于结合干扰研究，以确定RNase P RNA和tRNA的结构元件，是形成一个特定的RNase P RNA-tRNA复合物的关键。
We have studied the interaction of 3'-end variants of a (pre-)tRNA(Gly) with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus. To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNA(Gly) variants to RNase P RNAs was studied by gel retardation. A newly developed assay, based on the reduction of Pb2+-hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNA(Gly) variants. For E, coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNA(Gly) binding by 1 to 1.5 kcal/mol. Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3'-end-mediated interaction with RNase P RNA does not preferentially destabilize the transition state. Our data suggest another kinetic step following initial substrate binding to E. coli RNase P RNA (possibly a conformational rearrangement). For T. thermophilus RNase P RNA, product release of wild-type tRNA(Gly) CCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained with E. coli RNase P RNA. This supports the notion that a high-affinity binding site for the tRNA 3'-end is a ubiquitous feature of eubacterial P RNAs. Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.