biostudies-literatureEl Sayyed HWellcome TrustBiotechnology and Biological Sciences Research Council926-937.e4https://www.ebi.ac.uk/biostudies/studies/S-EPMC7618293biostudies-literatureUnknown84(5)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.Molecular cellSingle-molecule tracking reveals the functional allocation, in vivo interactions, and spatial organization of universal transcription factor NusG.PMC7618293226662BB/X015637/1BB/S008896/1BB/N018656/1091911107457226662/Z/22/Z110164/Z/15/Z204684/Z/16/Z224212/Z/21/ZEl Sayyed HGottesman MEStracy MKapanidis ANPambos OJfalseSingle-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.2024-01-01T00:00:00Z2024 Mar2026-06-23T03:12:10.212Z2026-06-23T03:09:37.748ZS-EPMC76182933838746110.1016/j.molcel.2024.01.025