Structural origins of Escherichia coli RNA polymerase open promoter complex stability

Significance The modulation of the rate of formation and of the lifetime of transcription initiation complexes is a critical point in gene expression control. In Escherichia coli, single-nucleotide changes can change the half-life of an RNA polymerase (RNAP)–promoter DNA complex by more than an order of magnitude. The origins of these effects are poorly understood. Using cryoelectron microscopy, we find that small alterations in the sequence or size of the transcription bubble trigger global changes in RNAP–DNA interactions and in DNA base stacking. Our results reveal that nonadditive structural changes allow a few crucial DNA positions to tune the transcription initiation complex lifetime from seconds to hours, influencing the rate and efficiency of the initial steps of RNA synthesis. The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA–RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand “scrunches” inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.