Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as 'transcriptional valves' which can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly we construct 1183 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to simultaneously characterize the entire pool at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate their function using nearby sequence context. This work provides new avenues for controlling transcription and demonstrates the value of long-read sequencing for exploring complex sequence-function landscapes.

中文翻译：

---

使用直接RNA测序对工程转录本亚型进行大规模平行表征

转录终止子发出信号，指示转录的RNA聚合酶（RNAP）应当终止并与DNA分离。但是，由于终止是随机的，因此可以生成两种不同形式的成绩单：一种终止于终止符，另一种贯穿阅读。控制这些转录异构体的丰度的能力将为生物工程师提供一种在转录水平上调节多基因构建体的机制。在这里，我们通过将终止子重新定位为“转录阀门”来探索这种可能性，该转录阀门可以调节RNAP读通的比例。使用一锅组合DNA装配，我们构建了T183 RNAP的1183个转录阀，并展示了如何基于纳米孔的直接RNA测序（dRNA-seq）可以在体外以核苷酸分辨率同时表征整个池，并阐明基因设计原理以使用附近的序列上下文调整和隔离其功能。这项工作提供了控制转录的新途径，并证明了长读测序对探索复杂的序列功能态势的价值。