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首页> 外文期刊>Biochemistry >Sequence specificity of a group II intron ribozyme: Multiple mechanisms for promoting unusually high discrimination against mismatched targets
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Sequence specificity of a group II intron ribozyme: Multiple mechanisms for promoting unusually high discrimination against mismatched targets

机译：II组内含子核酶的序列特异性：促进针对错配靶标的异常高区分度的多种机制

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摘要

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 (k(cat)/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 k(cat)/K-m value was compared to individual k(cat)/K-m 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 1 discrimination index (1/relative k(cat)/K-m), resulting in values > 10 000 in several cases. The Delta Delta G double dagger 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 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 (greater than or equal to 13 nucleotides). [References: 68]
机译：将II组内含子ai5γ重建为多周转核酶，可有效地反式切割小的寡核苷酸底物。由于可以获得二阶速率常数（k（cat）/ K-m或特异性常数）并将其与突变底物的值以及与其他核酶的值进行比较，因此该构建体使得研究序列特异性成为可能。本研究中使用的核酶由顺式连接的内含子域1和3，以及作为单独的催化辅因子的域5组成。这种核酶具有类似于反向剪接第一步的机制特征，其中套索内含子攻击外源RNA和DNA底物，因此，它可作为II组内含子迁移性序列特异性的模型。为了定量评估该核酶的序列特异性，将WT k（cat）/ Km值与单个包含碱基突变的一系列突变底物和核酶的单个k（cat）/ Km值进行比较，这些突变底物和核酶被设计为在沿两个核酶-底物识别螺旋的不同位置，这些错配具有明显的1区分指数（1 /相对k（cat）/ Km），在某些情况下导致值> 10000。取决于失配及其位置，失配的Delta Delta G双匕首范围为2至6 kcal / mol。核酶的高特异性归因于对双链体稳定性的影响（1-3 kcal / mol）和意想不到的对化学反应步骤的较大影响（0.5-2.5 kcal / mol）。此外，底物缔合伴随着一种能降低核酶与底物之间总结合能的能量，从而导致解离速率快于催化速率，并导致对长靶序列的切割具有很高的特异性（大于或等于13个核苷酸）。 [参考：68]

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