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.