Single-molecule experiments reveal NusG displaces transcription initiation factor σ70 from mature elongation complexes

The Escherichia coli transcription initiation factor σ70 was long believed to dissociate from RNA polymerase after promoter escape, following synthesis of short ~9-11 nt RNAs. However, recent evidence indicates σ70 is retained in elongation complexes (ECs), where it enhances pausing-implying that stochastic or factor-mediated displacement is required for processive elongation. Using fluorescence correlation spectroscopy, we quantified σ70 retention in the presence of elongation factors NusG and NusA, demonstrating that NusG-but not NusA-actively promotes σ70 dissociation. Single-molecule TIRF further revealed that NusG-mediated σ70 dissociation becomes favourable as ECs progress, with lower energy barriers for displacement in later elongation stages. These findings suggest a broader mechanistic paradigm in which elongation factors actively displace initiation factors to regulate transcription-a process that may be conserved across all domains of life.