Xiang Q, Qin P Z, Michels W J, Freeland K, Pyle A M
Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032, USA.
Biochemistry. 1998 Mar 17;37(11):3839-49. doi: 10.1021/bi972661n.
Group II intron ai5 gamma was reconstructed into a multiple-turnover ribozyme that efficiently cleaves small oligonucleotide substrates in-trans. This construct makes it possible to investigate sequence specificity, since second-order rate constants (kcat/K(m), or the specificity constant) can be obtained and compared with values for mutant substrates and with other ribozymes. The ribozyme used in this study consists of intron domains 1 and 3 connected in-cis, together with domain 5 as a separate catalytic cofactor. This ribozyme has mechanistic features similar to the first step of reverse-splicing, in which a lariat intron attacks exogenous RNA and DNA substrates, and it therefore serves as a model for the sequence specificity of group II intron mobility. To quantitatively evaluate the sequence specificity of this ribozyme, the WT kcat/Km value was compared to individual kcat/Km values for a series of mutant substrates and ribozymes containing single base changes, which were designed to create mismatches at varying positions along the two ribozyme-substrate recognition helices. These mismatches had remarkably large effects on the discrimination index (1/relative kcat/K(m)), resulting in values > 10,000 in several cases. The delta delta G++ for mismatches ranged from 2 to 6 kcal/mol depending on the mismatch and its position. The high specificity of the ribozyme is attributable to effects on duplex stabilization (1-3 kcal/mol) and unexpectedly large effects on the chemical step of reaction (0.5-2.5 kcal/mol). In addition, substrate association is accompanied by an energetic penalty that lowers the overall binding energy between ribozyme and substrate, thereby causing the off-rate to be faster than the rate of catalysis and resulting in high specificity for the cleavage of long target sequences (> or = 13 nucleotides).
II组内含子ai5γ被重构为一种多周转核酶，它能有效地在反式作用下切割小的寡核苷酸底物。这种构建体使得研究序列特异性成为可能，因为可以获得二级速率常数（kcat/K(m)，即特异性常数），并与突变底物的值以及其他核酶的值进行比较。本研究中使用的核酶由顺式连接的内含子结构域1和3以及作为单独催化辅因子的结构域5组成。这种核酶具有与反向剪接第一步相似的机制特征，在反向剪接中，套索状内含子攻击外源RNA和DNA底物，因此它可作为II组内含子移动性序列特异性的模型。为了定量评估这种核酶的序列特异性，将野生型kcat/Km值与一系列突变底物和含有单碱基变化的核酶的单个kcat/Km值进行比较，这些突变底物和核酶被设计为在两个核酶-底物识别螺旋的不同位置产生错配。这些错配对鉴别指数（1/相对kcat/K(m)）有显著的大影响，在几种情况下导致值>10,000。错配的ΔΔG++范围为2至6千卡/摩尔，具体取决于错配及其位置。核酶的高特异性归因于对双链体稳定性的影响（1 - 至3千卡/摩尔）以及对反应化学步骤的意外大影响（0.5 - 2.5千卡/摩尔）。此外，底物结合伴随着能量惩罚，这降低了核酶与底物之间的整体结合能，从而使解离速率比催化速率更快，并导致对长靶序列（≥13个核苷酸）切割具有高特异性。
