Hardt W D, Schlegl J, Erdmann V A, Hartmann R K
Institut für Biochemie, Freie Universität Berlin, FRG.
J Mol Biol. 1995 Mar 24;247(2):161-72. doi: 10.1006/jmbi.1994.0130.
We have studied the interaction of 3'-end variants of a (pre-)tRNAGly 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 tRNAGly variants to RNase P RNAs was studied by gel retardation. A newly developed assay, based on the reduction of Pb(2+)-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-tRNAGly variants. For E. coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGly 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 tRNAGly 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.
我们研究了（前体）甘氨酰tRNA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之间的相互作用。为了从整体催化反应中剖析tRNA结合的热力学，通过凝胶阻滞实验研究了成熟甘氨酰tRNA变体与RNase P RNA的特异性结合。利用一种新开发的检测方法，该方法基于由于tRNA与RNase P RNA形成复合物而导致CCA末端Pb(2+)水解减少，来确定解离常数。通过对相应前体甘氨酰tRNA变体的单周转和多周转动力学分析对结合数据进行了补充。对于大肠杆菌RNase P RNA，获得了以下结果。用pCp或三个核苷酸（AUA）延伸CCA可使凝胶分辨的甘氨酰tRNA结合稳定1至1.5千卡/摩尔。将CCA中的第一个C替换为A、G或U会导致结合亲和力降低100倍以上，这相当于结合能损失3.5至4.5千卡/摩尔。然而，单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丧失不会优先使过渡态不稳定。我们的数据表明，在初始底物与大肠杆菌RNase P RNA结合之后还有另一个动力学步骤（可能是构象重排）。对于嗜热栖热菌RNase P RNA，野生型甘氨酰tRNA CCAAUA的产物释放在多周转反应中不是限速步骤。然而，CCA突变的影响与大肠杆菌RNase P RNA的情况相似。这支持了这样一种观点，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，这里获得的结果进一步证明凝胶阻滞实验适用于结合干扰研究，以鉴定对形成特定的RNase P RNA - tRNA复合物至关重要的RNase P RNA和tRNA的结构元件。
