Leistra Abigail N, Amador Paul, Buvanendiran Aishwarya, Moon-Walker Alex, Contreras Lydia M
McKetta Department of Chemical Engineering, University of Texas at Austin , 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States.
Microbiology Graduate Program, University of Texas at Austin , 100 E. 24th Street Stop A6500, Austin, Texas 78712, United States.
ACS Synth Biol. 2017 Dec 15;6(12):2228-2240. doi: 10.1021/acssynbio.7b00185. Epub 2017 Aug 10.
Bacterial small RNAs (sRNAs) have been established as powerful parts for controlling gene expression. However, development and application of engineered sRNAs has primarily focused on regulating novel synthetic targets. In this work, we demonstrate a rational modular RNA engineering approach that uses in vivo structural accessibility measurements to tune the regulatory activity of a multisubstrate sRNA for differential control of its native target network. Employing the CsrB global sRNA regulator as a model system, we use published in vivo structural accessibility data to infer the contribution of its local structures (substructures) to function and select a subset for engineering. We then modularly recombine the selected substructures, differentially representing those of presumed high or low functional contribution, to build a library of 21 CsrB variants. Using fluorescent translational reporter assays, we demonstrate that the CsrB variants achieve a 5-fold gradient of control of well-characterized Csr network targets. Interestingly, results suggest that less conserved local structures within long, multisubstrate sRNAs may represent better targets for rational engineering than their well-conserved counterparts. Lastly, mapping the impact of sRNA variants on a signature Csr network phenotype indicates the potential of this approach for tuning the activity of global sRNA regulators in the context of metabolic engineering applications.
细菌小RNA（sRNA）已被确立为控制基因表达的有力元件。然而，工程化sRNA的开发与应用主要集中在调控新型合成靶点上。在这项工作中，我们展示了一种合理的模块化RNA工程方法，该方法利用体内结构可及性测量来调节多底物sRNA的调控活性，以对其天然靶标网络进行差异控制。以CsrB全局sRNA调节因子作为模型系统，我们利用已发表的体内结构可及性数据来推断其局部结构（子结构）对功能的贡献，并选择一个子集进行工程改造。然后，我们模块化地重组所选子结构，以不同方式呈现那些假定具有高或低功能贡献的子结构，构建了一个包含21个CsrB变体的文库。通过荧光翻译报告分析，我们证明CsrB变体对特征明确的Csr网络靶标实现了5倍的调控梯度。有趣的是，结果表明，在长的多底物sRNA中，保守性较低的局部结构可能比保守性高的局部结构更适合作为合理工程改造的靶点。最后，绘制sRNA变体对标志性Csr网络表型的影响，表明了这种方法在代谢工程应用背景下调节全局sRNA调节因子活性的潜力。
