Exploring RNA polymerase dynamics and regulatory architecture across divergent bacterial species

The National Institute of General Medical Sciences awarded The University of Texas Southwestern Medical Center $456,500 on August 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to explore RNA polymerase dynamics and regulatory architecture across divergent bacterial species.

The recipient will develop and apply a new sequencing platform that enables purified RNA polymerase to transcribe entire bacterial genomes under defined biochemical conditions, with subsequent isolation and high-throughput sequencing of RNA 3' ends to map pause and termination sites at single-nucleotide resolution. This platform is designed to overcome limitations of in vivo approaches by bypassing confounding cellular networks and will be adaptable across multiple bacterial species. The research focuses on mechanisms of RNA polymerase pausing and termination, with the long-term goal of identifying species-specific differences in transcriptional regulation that could inform development of species-specific RNA polymerase inhibitors as research tools and antimicrobial leads.

Work is performed in Dallas, Texas. The period of performance runs through March 31, 2031.

Abstract

Transcription, the first step in gene expression, is carried out by RNA polymerase (rnap). Rnap is a multi- subunit protein that transcribes DNA into RNA in a tightly regulated process of transcription. In bacteria, the catalytic core of rnap is conserved, yet distinct modules and peripheral subunits confer species-specific modes of regulation. A deeper understanding of rnap structure, function, and regulation promises to rationally manipulate rnap and gene expression, while also yielding insights applicable to eukaryotic systems. Our current knowledge of bacterial transcription mechanisms is largely derived from the model organism e. Coli. However, IT has become increasingly evident that the process of transcription is more complex in various bacteria, and even a species closely related to e. Coli exhibits markedly different regulation of transcription. The long-term goal of my research is to study how the mechanisms of transcription and its regulation differ across bacterial species and to exploit these differences for species-specific rnap inhibitors as research tools and antimicrobial leads. In this proposal, i focus on two fundamental processes: rnap pausing and termination. Genome-wide studies of rnap pausing (net-SEQ) and termination (term-SEQ) have provided valuable insights, but these in vivo approaches are confounded by the complex cellular networks that obscure direct mechanistic contributions. To overcome this challenge, we have developed a new sequencing platform that bypasses the limitations of in vivo approaches. In our system, purified rnap transcribes entire bacterial genomes under defined biochemical conditions. We then isolate and high-throughput sequence RNA 3′ ends, enabling the precise mapping of pause and termination sites at single-nucleotide resolution. Importantly, this platform is readily adaptable to many bacterial species and provides a controlled environment to dissect how specific factors influence transcriptional pausing and termination. With this new approach in combination with cryo-EM, biochemical assays, deep- learning and bacterial genetics, we will investigate rnap pausing and termination across phylogenetically distant bacterial species from three perspectives: (1) intrinsic properties of rnap, (2) the sequence and structural features of genomic DNA, and (3) transcriptional factors that modulate rnap activity. We predict that the interplay among these elements collectively shapes transcriptional outcomes. In addition, our in vitro sequencing platform has revealed premature termination events in 5′ untranslated regions (5′utrs) that do not correspond to any known riboswitches. Riboswitches are structured RNA elements within 5′utrs that bind specific nutrient ligands to control downstream gene expression. Intriguingly, all of the premature termination sites we identified are associated with stable RNA secondary structures, suggesting previously unrecognized regulatory roles. We will further dissect the mechanisms and functional impact of these events. Together, our studies will uncover fundamental principles of transcription, identify new opportunities for antibiotic development, and inform synthetic biology applications.