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Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing

Matt Tarnowski

Matt Tarnowski, University of Bristol, UK

Abstract

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should disassociate from DNA. Two forms of transcript can arise: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms allows regulation of multi-gene constructs. We repurpose terminators as ‘transcriptional valves’ which can tune the proportion of read-through. We show how nanopore-based direct RNA sequencing simultaneously characterizes 1,183 transcriptional valves for T7 RNAP at nucleotide resolution in vitro and unravels their genetic design principles. This work elucidates a new mechanism to control transcription and demonstrates how long-read sequencing can reveal sequence-function landscapes.

Bio

Matt Tarnowski is a Ph.D. student at the University of Bristol researching how microorganisms work by looking at their genetic code. He has growing interests in fermenting, foraging, regenerative agroecology, and responsible research.