Mechanisms of transcription pausing and fidelity in prokaryotes and eukaryotes

Division Of Basic Sciences - Nci

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Abstract

Transcriptional regulation by RNAP pausing and termination plays a pivotal role in gene expression across bacterial, yeast, and mammalian systems. In eukaryotes, promoter-proximal pausing is essential for modulating transcription initiation, alternative splicing, RNA processing, termination, and transcription-coupled DNA repair. In prokaryotes, pausing critically influences gene regulation by riboswitches, promoter escape kinetics, coupled transcription-translation, and mechanisms of intrinsic termination and antitermination. Building on decades of research, my laboratory has systematically investigated how specific cis-acting signals in DNA and nascent RNA and trans-acting Nus/Gre/Rho/N protein factors influence pausing and termination. We focus on different states of RNAP, the elemental, pre-translocation, and backtracked pauses in E. coli and B. subtilis. Using advanced sequencing technologies (NET-seq, RNET-seq, D-RNET-seq, TNET-seq, Term-seq) and engineered bacterial strains carrying conditional knockouts/knockdowns of general elongation factors, we validate these regulatory mechanisms in vitro and in vivo. My lab developed TNET-seq and D-RNET-seq for mapping the original 5-prime ends of nascent RNA, and for simultaneous sequencing of both the transcribed DNA and nascent RNA (RNAP/Pol II footprints on nascent RNA and transcribed DNA) from isolated transcription complexes, respectively. A key component of our current work examines how mis-regulated transcription pausing impacts physiology and gene expression in human cancer cell lines including TNBC, Non-TNBC, HEK293, and HeLa cells. Dysregulation at the level of Pol II pausing, alternative splicing, and 3-prime RNA processing can contribute to cancer progression, notably in breast cancer. Leveraging genome-wide NET-seq, RNET-seq, RNA-seq, ChIP-seq, and nucleosome profiling, we investigate Pol II pausing in TNBC, non-TNBC, HeLa, and HEK293 cell lines. Emphasis is placed on Pol II backtracking, transcript cleavage factors (TFIIS, Spt5), and termination factors (XRN2), including their roles in promoter escape and termination downstream of poly(A) sites (PAS). For functional validation, we employ shRNA knockdown and CRISPR knockout strategies to dissect the roles of these regulatory elements in normal and stress conditions. By integrating bacterial and eukaryotic research, our lab seeks to elucidate the universal and divergent strategies of RNAP-mediated transcriptional control, contributing both fundamental insights and potential therapeutic avenues.