Christian E L, Harris M E
Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Biochemistry. 1999 Sep 28;38(39):12629-38. doi: 10.1021/bi991278a.
The ribonuclease P (RNase P) ribozyme is an endonuclease that binds precursor tRNAs and catalyzes the removal of 5' leader nucleotides. Biochemical and photo-cross-linking studies have identified sites of contact between the mature tRNA domain of pre-tRNA and the ribozyme; however, relatively little is known about the location of the 5' leader in the ribozyme-substrate complex. To investigate the local three-dimensional environment of the 5' leader, we employed the short-range photo-cross-linking agent 4-thiouridine (s(4)U). The s(4)U photoagent was incorporated into a series of pre-tRNA substrates containing unique uridine residues in the 5' leader sequence at positions -1, -3, -5, -7, or -10. The modified substrates formed high-affinity complexes with the ribozyme and produced discrete intermolecular cross-links to RNase P RNA from Bacillus subtilis. Locations of the cross-linked nucleotides in the ribozyme and pre-tRNA were determined by reverse transcriptase primer extension. Photoagents incorporated into the 5' leader detected discrete elements of ribozyme structure in a progression from J18/2 to L15 to P3. Importantly, all of the cross-linked species retained the ability to cleave the covalently attached pre-tRNA, indicating that the cross-links reflect the native structure of the ribozyme-substrate complex. Together with available structural and biochemical data, the cross-linking results suggest a model for the position of the 5' leader within the ground-state ribozyme-substrate complex.
核糖核酸酶P（RNase P）核酶是一种内切核酸酶，它结合前体tRNA并催化去除5'前导核苷酸。生化和光交联研究已经确定了前体tRNA的成熟tRNA结构域与核酶之间的接触位点；然而，关于5'前导序列在核酶-底物复合物中的位置，人们了解得相对较少。为了研究5'前导序列的局部三维环境，我们使用了短程光交联剂4-硫尿苷（s(4)U）。将s(4)U光试剂掺入一系列前体tRNA底物中，这些底物在5'前导序列中-1、-3、-5、-7或-10位置含有独特的尿苷残基。修饰后的底物与核酶形成高亲和力复合物，并与来自枯草芽孢杆菌的RNase P RNA产生离散的分子间交联。通过逆转录酶引物延伸确定核酶和前体tRNA中交联核苷酸的位置。掺入5'前导序列的光试剂从J18/2到L15再到P3逐步检测到核酶结构的离散元件。重要的是，所有交联物种都保留了切割共价连接的前体tRNA的能力，这表明交联反映了核酶-底物复合物的天然结构。结合现有的结构和生化数据，交联结果提出了一个关于5'前导序列在基态核酶-底物复合物中位置的模型。
