The stringent response to nutrient deprivation is a stress response found throughout the bacterial domain of life. Although first described in proteobacteria for matching ribosome synthesis to the cell's translation status and for preventing formation of defective ribosomal particles, the response is actually much broader, regulating many hundreds of genes-some positively, some negatively. Utilization of the signaling molecules ppGpp and pppGpp for this purpose is ubiquitous in bacterial evolution, although the mechanisms employed vary. In proteobacteria, the signaling molecules typically bind to two sites on RNA polymerase, one at the interface of the β' and ω subunits and one at the interface of the β' secondary channel and the transcription factor DksA. The β' secondary channel is targeted by other transcription regulators as well. Although studies on the transcriptional outputs of the stringent response date back at least 50 years, the mechanisms responsible are only now coming into focus.

Highlights d Cryo-EM structures of 7 intermediates in promoter opening pathway from RPc to RPo d Intermediates populated by using an inhibitor and a promoter with unstable RPo d RNAP and DNA conformational changes in mobile regions mark the steps in the pathway d Transient interactions identified in intermediates are not found in RPc or RPo

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.

The Escherichia coli F element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.

σ is an alternative sigma factor, encoded by the gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σ general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σ levels transcriptionally, translationally, and post-translationally in It was shown previously that the transcription factor DksA and its cofactor ppGpp are among the many factors governing σ synthesis, thus playing an important role in activation of the σ stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σ levels. In addition, based on effects of DksA, a previously identified DksA variant with increased affinity for RNAP, we show that DksA can increase σ activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σ and also increase transcription from other promoters, including P and P By improving the translation and stabilization of σ, as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation. Bacteria spend relatively little time in log phase outside of the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σ Understanding the factors governing σ activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σ levels.