Crary S M, Niranjanakumari S, Fierke C A
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 1998 Jun 30;37(26):9409-16. doi: 10.1021/bi980613c.
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-tRNAAsp 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 >/=15-fold compared to tRNAAsp 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-tRNAAsp 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-tRNAAsp and the protein component of RNase P.
核糖核酸酶P（RNase P）是一种核糖核蛋白复合物，可催化tRNA成熟5'端的形成。为了研究蛋白质成分在增强枯草芽孢杆菌RNase P与底物亲和力方面的作用（Kurz, J. C., Niranjanakumari, S., Fierke, C. A. (1998) Biochemistry 37, 2393），我们分析了含有不同长度（1 - 33个核苷酸）5'前导序列的前体tRNAAsp底物的结合和切割动力学及热力学。这些数据表明，全酶催化的切割速率常数不依赖于前导序列长度；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，这反映在底物亲和力提高了多达4千卡/摩尔。特别是，RNase P的蛋白质成分稳定了与前体tRNA底物5'前导序列中 - 2和 - 5位核苷酸的相互作用。与tRNAAsp相比，1个核苷酸的前导序列由于酶 - 底物复合物的基态不稳定而使底物亲和力降低≥15倍。由于P蛋白稳定的P RNA - 前体tRNA接触，将前导序列长度增加到2个核苷酸可克服这种不稳定。含有5个核苷酸前导序列的底物进一步增强了RNase P全酶（而非单独的RNA）对前体tRNAAsp的亲和力。这些数据表明，前体tRNAAsp的5'前导序列与RNase P的蛋白质成分之间发生了新的直接或间接相互作用。
