Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp
Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp
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DOI：
10.1021/bi972530m
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发表时间：
1998-02-24
期刊：
影响因子：
2.9
通讯作者：
Fierke, CA
中科院分区：
文献类型：
--
作者：
Kurz, JC;Niranjanakumari, S;Fierke, CA
核糖核酸酶P （RNase P）是一种切割前体tRNA形成成熟tRNA 5'端的内切酶，由催化RNA亚基和小蛋白亚基组成。通过稳态动力学、瞬态动力学和配体亲和测量，研究了枯草芽孢杆菌RNase P蛋白组分在催化枯草芽孢杆菌前体tRNA（Asp）裂解中的功能，并比较了RNase P全酶与RNase P RNA在10 mM MgCl2、100 mM NH4Cl中的功能特性。蛋白质成分适度地影响了几个步骤，包括tRNA解离速率常数、tRNA亲和力和磷酸二酯键裂解速率常数的小于或等于10倍的增加。然而，该蛋白主要影响底物结合，将RNase P对预trna （Asp）的亲和力提高了10(4)倍，这是通过10 mM MgCl2测量的预trna （Asp）解离率和结合速率常数以及10 mM CaCl2测量的结合等温线来确定的，使用凝胶过滤分离酶结合和游离的预trna （Asp）。因此，RNase P中蛋白质组分的主要作用是通过增强酶与底物5'前体段的相互作用来促进对pre-tRNA的识别，而不是像蛋白质促进的I组内含子自剪接那样稳定折叠RNA的第三级结构。此外，蛋白质组分在生理条件下最大限度地提高了RNase P的效率，并将产物抑制降到最低。
Ribonuclease P (RNase P) is an endonuclease that cleaves precursor tRNA to form the 5'-end of mature tRNA and is composed of a catalytic RNA subunit and a small protein subunit. The function of the protein component of Bacillus subtilis RNase P in catalysis of B. subtilis precursor tRNA(Asp) cleavage has been elucidated using steady-state kinetics, transient kinetics, and ligand affinity measurements to compare the functional properties of RNase P holoenzyme to RNase P RNA in 10 mM MgCl2, 100 mM NH4Cl. The protein component modestly affects several steps including less than or equal to 10-fold increases in the rate constant for tRNA dissociation, the affinity of tRNA, and the rate constant for phosphodiester bond cleavage. However, the protein principally affects substrate binding, increasing the affinity of RNase P for pre-tRNA(Asp) by a factor of 10(4) as determined from both the ratio of the pre-tRNA(Asp) dissociation and association rate constants measured in 10 mM MgCl2 and a binding isotherm measured in 10 mM CaCl2 using gel filtration to separate enzyme-bound and free pre-tRNA(Asp). Therefore, the main role of the protein component in RNase P is to facilitate recognition of pre-tRNA by enhancing the interaction between the enzyme and the 5'-precursor segment of the substrate, rather than stabilizing the tertiary structure of the folded RNA as has been observed for protein-facilitated group I intron self-splicing, Furthermore, the protein component maximizes the efficiency of RNase P under physiological conditions and minimizes product inhibition.