Abstract
TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.
Highlights
Transcription initiation is a major control point for gene expression
TraR-like proteins are widespread in proteobacteria and related bacteriophage and plasmids (Gopalkrishnan et al, 2017; Gourse et al, 2018)
While the in vivo function of TraR is incompletely understood, TraR engages with RNA polymerase (RNAP) in much the same way as DksA/ppGpp, utilizing the same residues in the b'rim-helices that contribute to ppGpp site 2 in the DksA-ppGpp-RNAP complex, and uses its N-terminal a-helix to bind in the RNAP secondary channel near the RNAP active site (Gopalkrishnan et al, 2017; Ross et al, 2016)
Summary
Transcription initiation is a major control point for gene expression. In bacteria, a single catalytically active RNA polymerase (RNAP) performs all transcription, but a σ factor is required for promoter utilization (Burgess et al, 1969; Feklistov et al, 2014). Bacterial RNAP-binding factors, encoded by the chromosome or by bacteriophage or extrachromosomal elements, interact with several different regions of the enzyme to regulate its functions (Haugen et al, 2008). One such factor is ppGpp, a modified nucleotide that functions together with the RNAP-binding protein DksA in Eco to reprogram bacterial metabolism in response to nutritional stresses during the so-called stringent response. PpGpp and DksA alter the expression of as many as 750 genes within 5 minutes of ppGpp induction (Paul et al, 2004a; 2005; Sanchez-Vazquez et al, 2019), inhibiting, for example, promoters responsible for ribosome biogenesis and activating promoters responsible for amino acid synthesis
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