Department of Genetics, Stanford University, Stanford, CA, 94305, USA.
Department of Biochemistry, Stanford University, Stanford, CA, 94305, USA.
Nat Commun. 2020 Apr 3;11(1):1663. doi: 10.1038/s41467-020-15540-1.
Massively parallel, quantitative measurements of biomolecular activity across sequence space can greatly expand our understanding of RNA sequence-function relationships. We report the development of an RNA-array assay to perform such measurements and its application to a model RNA: the core glmS ribozyme riboswitch, which performs a ligand-dependent self-cleavage reaction. We measure the cleavage rates for all possible single and double mutants of this ribozyme across a series of ligand concentrations, determining k and K values for active variants. These systematic measurements suggest that evolutionary conservation in the consensus sequence is driven by maintenance of the cleavage rate. Analysis of double-mutant rates and associated mutational interactions produces a structural and functional mapping of the ribozyme sequence, revealing the catalytic consequences of specific tertiary interactions, and allowing us to infer structural rearrangements that permit certain sequence variants to maintain activity.
大规模平行、定量测量生物分子在序列空间中的活性,可以极大地扩展我们对 RNA 序列-功能关系的理解。我们报告了一种 RNA 微阵列测定法的开发及其在模型 RNA 上的应用:核心 glmS 核酶核糖开关,它进行配体依赖性自我切割反应。我们测量了该核酶在一系列配体浓度下所有可能的单突变体和双突变体的切割率,确定了活性变体的 k 和 K 值。这些系统测量表明,共识序列中的进化保守性是由切割率的维持所驱动的。对双突变体速率和相关突变相互作用的分析产生了核酶序列的结构和功能映射,揭示了特定三级相互作用的催化后果,并使我们能够推断出允许某些序列变体保持活性的结构重排。