Department of Cell and Molecular Biology, Box 596, Uppsala University, SE-751 24 Uppsala, Sweden, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA and Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, SE-751 24 Uppsala, Sweden.
Nucleic Acids Res. 2014 Jan;42(1):631-42. doi: 10.1093/nar/gkt853. Epub 2013 Oct 3.
We have used model substrates carrying modified nucleotides at the site immediately 5' of the canonical RNase P cleavage site, the -1 position, to study Escherichia coli RNase P RNA-mediated cleavage. We show that the nucleobase at -1 is not essential but its presence and identity contribute to efficiency, fidelity of cleavage and stabilization of the transition state. When U or C is present at -1, the carbonyl oxygen at C2 on the nucleobase contributes to transition-state stabilization, and thus acts as a positive determinant. For substrates with purines at -1, an exocyclic amine at C2 on the nucleobase promotes cleavage at an alternative site and it has a negative impact on cleavage at the canonical site. We also provide new insights into the interaction between E. coli RNase P RNA and the -1 residue in the substrate. Our findings will be discussed using a model where bacterial RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal(II)-activated H2O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.
我们使用了在标准 RNase P 切割位点 5' 处立即携带修饰核苷酸的模型底物，即-1 位，来研究大肠杆菌 RNase P RNA 介导的切割。我们表明，-1 位的核碱基不是必需的，但它的存在和身份有助于提高效率、切割保真度和过渡态的稳定性。当 -1 位存在 U 或 C 时，核碱基上 C2 的羰基氧有助于过渡态稳定，因此是一个正决定因素。对于 -1 位为嘌呤的底物，核碱基上 C2 的环外胺促进在替代位点的切割，并且对在标准位点的切割有负面影响。我们还提供了大肠杆菌 RNase P RNA 与底物中 -1 位残基相互作用的新见解。我们的发现将使用一种模型进行讨论，其中细菌 RNase P 切割通过构象辅助机制进行，该机制将金属(II)激活的 H2O 定位在直链攻击磷原子上，导致磷酸二酯键断裂。
