RNAP-seq: in vitro genome-scale transcription reveals preferential RNA polymerase pausing on Clostridioides difficile antisense DNA

In vivo analyses using high-throughput DNA sequencing readouts (e.g., RNA-seq, Term-seq, and NET-seq) have revealed that transcriptional pausing by RNA polymerase (RNAP) underpins the regulation of gene transcription. However, these in vivo methods are difficult to apply in non-model organisms and lack the power of bottom-up biochemical analysis that can deconvolute the confounding effects of the cellular environment.

Here we report a method for unbiased transcription of entire genomes in vitro called RNAP-seq, which is capable of defining pause, arrest, and termination sites with single-nucleotide resolution and of defining the regulatory contributions of cis-acting RNA and DNA sequences and dissociable transcription factors with complete control over transcription conditions. Using RNAP-seq to compare transcription by recombinant RNAPs from Clostridioides difficile (Cdf) and Escherichia coli (Eco), we discovered that Cdf RNAP exhibits pausing signatures distinct from Eco RNAP.