ABSTRACT

Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) is known to respond to two types of termination signals, but the mechanism underlying such termination, especially the specific elements of the polymerase involved, is still unclear, due to a lack of knowledge with respect to the structure of the termination complex. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation (S43Y) that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNAP (RdRp) activity of T7 RNAP without impeding the major DNA-dependent RNAP (DdRp) activity of the enzyme. S43 is located in a hinge region and regulates the transformation between transcription initiation and elongation of T7 RNAP. Steady-state kinetics analysis and an RNA binding assay indicate that the S43Y mutation increases the transcription efficiency while weakening RNA binding of the enzyme. As an enzymatic reagent for in vitro transcription, the T7 RNAP S43Y mutant reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

 抽象的

转录终止是人们最不了解的基因表达过程之一。作为转录研究的原型模型，单亚基 T7 RNA 聚合酶 (RNAP) 已知可响应两种类型的终止信号，但这种终止的机制，特别是所涉及的聚合酶的特定元件仍不清楚，因为缺乏对终止复合体结构的了解。在这里，我们应用噬菌体辅助的连续进化来获得 T7 RNAP 的变体，该变体可以绕过具有茎环结构的典型 I 类 T7 终止子。通过体内选择和体外表征，我们发现一个单一突变 (S43Y) 显着降低了所有测试的转录终止子上 T7 RNAP 的终止效率。巧合的是，S43Y 突变几乎消除了 T7 RNAP 的 RNA 依赖性 RNAP (RdRp) 活性，但不妨碍该酶的主要 DNA 依赖性 RNAP (DdRp) 活性。 S43 位于铰链区，调节 T7 RNAP 的转录起始和延伸之间的转化。稳态动力学分析和 RNA 结合测定表明，S43Y 突变提高了转录效率，同时削弱了酶的 RNA 结合。作为体外转录的酶试剂，T7 RNAP S43Y 突变体减少了径流 RNA 合成中不需要的终止，并产生具有更高末端同质性的 RNA。