Gene transcription is the process cells use to copy DNA instructions into RNA. This is one of the most important steps in gene regulation, because it helps determine when genes are turned on, how strongly they are expressed, and when transcription stops. A key player in this process is RNA polymerase, the enzyme that reads DNA and produces RNA.

Researchers UT Southwestern Medical Center have developed a method called RNAP-seq to study transcription across entire bacterial genomes in a controlled laboratory setting.

Traditional RNA sequencing methods such as RNA-seq, Term-seq, and NET-seq have shown that RNA polymerase pausing is an important part of gene regulation. However, these methods are often performed inside living cells, where many overlapping factors make it difficult to isolate the direct behavior of RNA polymerase. They can also be harder to apply in non-model organisms.

RNAP-seq takes a different approach. Instead of measuring RNA production inside a living cell, the method allows researchers to transcribe entire genomes in vitro, meaning under controlled laboratory conditions. This gives scientists more control over the transcription environment and lets them test how DNA sequence, RNA sequence, and added transcription factors influence RNA polymerase behavior.

Schematic workflow and scope of RNAP-seq

A, Schematic of the RNAP-seq workflow. RNAP-derived 3′ ends include signals arising from both transcriptional pausing and termination. Transcription termination sites (TTS) are identified and excluded from the full set of 3′ ends using a bioinformatic pipeline (Materials and Methods). B, Schematic illustrating RNAP-seq transcription of cognate genomic DNA (gDNA, top) and heterologous genomic DNA (bottom) by Eco and Cdf RNAP. C, Bar graph showing the fraction of TTS identified by RNAP-seq using Cdf RNAP on Cdf gDNA (orange) and Eco RNAP on Eco gDNA (blue), relative to in cellulo TTS datasets. If multiple termination sites identified by RNAP-seq fell within a ±5 nt window of a terminator in the corresponding in cellulo dataset, they were considered a single site. The total number of TTS detected by RNAP-seq for Cdf and Eco RNAP is indicated on the X-axis. D, Bar graph showing the fraction of intrinsic, Rho-dependent, and unclassified TTS identified by RNAP-seq using Eco RNAP on Eco gDNA, relative to the in cellulo dataset. The number of TTS detected by RNAP-seq in each category is indicated on the X-axis. E, Genome wide distribution of 3′ end halt signals on the Cdf genome. Outer circle: gene annotations; middle circle: 3′ end coverage by Cdf RNAP; inner circle: 3′ end coverage by Eco RNAP. Red and blue colors represent positive and negative strands, respectively. Mb, megabase.

Using RNAP-seq, the team compared RNA polymerases from Clostridioides difficile and Escherichia coli. These bacteria are biologically different, and their RNA polymerases showed distinct pausing patterns. The C. difficile enzyme paused more often and more strongly at T-rich DNA sequences, especially in antisense regions of genes.

The researchers also studied NusG, a transcription factor from C. difficile. RNAP-seq revealed that this factor acts preferentially in riboswitch control regions, which are RNA-based regulatory elements that help bacteria respond to changing conditions. This shows how RNAP-seq can be used not only to map RNA polymerase behavior, but also to understand how specific regulatory proteins shape transcription across a genome.

These findings suggest that genome sequence and RNA polymerase properties may evolve together. In other words, the DNA composition of an organism and the behavior of its transcription machinery can shape each other over time, influencing how genes are regulated.

RNAP-seq could be useful for studying many bacterial species, including organisms that are difficult to analyze with standard in vivo methods. By providing single-nucleotide resolution of transcriptional pausing, arrest, and termination, it offers a powerful way to explore the basic rules that govern bacterial gene expression.