The protein component of Bacillus subtilis ribonuclease P increases catalytic efficiency by enhancing interactions with the 5′ leader sequence of pre-tRNAAsp
The protein component of Bacillus subtilis ribonuclease P increases catalytic efficiency by enhancing interactions with the 5′ leader sequence of pre-tRNAAsp
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DOI：
10.1021/bi980613c
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发表时间：
1998-06-30
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：
Crary, SM;Niranjanakumari, S;Fierke, CA
核糖核酸酶P（RNase P）是一种核糖核蛋白复合物，催化tRNA成熟5'端的形成。为了研究蛋白质组分在增强枯草芽孢杆菌RNase P对底物的亲和力中的作用（Kurz，J.C.，Niranjanakumari，S.，菲尔克角A.（1998）Biochemistry 37，2393），分析了含有不同长度（1-33个核苷酸）的5'前导序列的前tRNA（Asp）底物的结合和切割的动力学和热力学。这些数据表明全酶催化的裂解速率常数不依赖于前导序列的长度;然而，底物与全酶结合的缔合速率常数随着前导序列长度的增加而增加，这反映在高达4 kcal/mol的增强的底物亲和力中。特别地，RNase P的蛋白质组分稳定与前体tRNA底物的5'前导序列中的-2和-5处的核苷酸的相互作用。由于酶-底物复合物的基态不稳定，与tRNA（Asp）相比，1个核苷酸的前导序列使底物亲和力降低大于或等于15倍。由于P蛋白稳定的P RNA-前-tRNA接触，通过将前导序列的长度增加到2个核苷酸来克服这种不稳定。RNase P全酶（而不是单独的RNA）对前体tRNA（Asp）的亲和力进一步增强含有5个核苷酸前导序列的底物。这些数据表明，新的直接或间接的相互作用发生在5'前导序列的前tRNA（Asp）和RNase P的蛋白质组分之间。
Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the formation of the mature 5' end of tRNA. To investigate the role of the protein component in enhancing the affinity of Bacillus subtilis RNase P for substrate (Kurz, J. C., Niranjanakumari, S., Fierke, C. A. (1998) Biochemistry 37, 2393), the kinetics and thermodynamics of binding and cleavage were analyzed for pre-tRNA(Asp) substrates containing 5' leader sequences of varying lengths (1-33 nucleotides). These data demonstrate that the cleavage rate constant catalyzed by the holoenzyme is not dependent on the leader length; however, the association rate constant for substrate binding to holoenzyme increases as the length of the leader increases, and this is reflected in enhanced substrate affinity of up to 4 kcal/mol. In particular, the protein component of RNase P stabilizes interactions with nucleotides at -2 and -5 in the 5' leader sequence of the pre-tRNA substrate. A 1 nucleotide leader decreases substrate affinity greater than or equal to 15-fold compared to tRNA(Asp) due to ground-state destabilization of the enzyme-substrate complex. This destabilization is overcome by increasing the length of the leader to 2 nucleotides due to P RNA-pre-tRNA contacts that are stabilized by the P protein. The affinity of RNase P holoenzyme (but not RNA alone) for pre-tRNA(Asp) is further enhanced with a substrate containing a 5 nucleotide leader. These data indicate that novel direct or indirect interactions occur between the 5' leader sequence of pre-tRNA(Asp) and the protein component of RNase P.