The interaction between σ⁷⁰ and the β-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation

The σ-subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when σ associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of σ and the flap domain of the RNAP β-subunit (β-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli σ⁷⁰ that have specific effects on the σ⁷⁰ region 4/β-flap interaction, either weakening or strengthening it. Using these σ⁷⁰ mutants, we demonstrate that the σregion 4/β-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the β-flap, σ region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of σ-core interactions.