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

Bacterial gene expression is highly regulated to allow cells to grow and adapt. Much regulation occurs during transcription elongation, where RNA polymerase (RNAP) extends nascent RNA transcripts aided by global and universally-conserved elongation factor NusG. NusG modulates transcription by inhibiting pausing and backtracking; promoting anti-termination on ribosomal RNA (rrn) operons; coupling transcription with translation on mRNA genes; and stimulating Rho-dependent termination on toxic genes. Despite extensive work on NusG, its functional allocation and spatial distribution in vivo is unknown. Here, we addressed these long-standing questions using single-molecule tracking and super-resolution imaging of NusG in live E. coli cells. We found that, under conditions of moderate growth, NusG is mainly present as a population that associates indirectly with the chromosome via RNAP in transcription elongation complexes, and a slowly diffusing population we identified as a NusG complex with the 30S ribosomal subunit; this complex offers a "30S-guided" path for NusG to enter transcription elongation. Only ~10% of total NusG was fast-diffusing, with the mobility of this population suggesting that free NusG interacts non-specifically with DNA for >50% of the time. Using antibiotics and deletion mutants, we showed that most chromosome-associated NusG is involved in rrn anti-termination and in transcriptiontranslation coupling. NusG involvement in rrn anti-termination was mediated via its participation in phase-separated transcriptional condensates. Our work illuminates the diverse activities of a central regulator while offering a guide on how to dissect the roles of multi-functional machines using in vivo imaging.