Single-molecule tracking reveals the functional allocation, in vivo interactions, and spatial organization of universal transcription factor NusG.

During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only ∼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging.

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Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of interests A.N.K. is a co-founder and shareholder of Oxford Nanoimaging (ONI), a company that builds, sells, and supports miniaturized fluorescence microscopes for single-molecule imaging, including for single-particle tracking.