Allosteric Effector ppGpp Potentiates the Inhibition of Transcript Initiation by DksA

Abstract

DksA and ppGpp are the central players in the stringent response and mediate a complete reprogramming of the transcriptome. A major component of the response is a reduction in ribosome synthesis, which is accomplished by the synergistic action of DksA and ppGpp bound to RNA polymerase (RNAP) inhibiting transcription of rRNAs. Here, we report the X-ray crystal structures of Escherichia coli RNAP in complex with DksA alone and with ppGpp. The structures show that DksA accesses the template strand at the active site and the downstream DNA binding site of RNAP simultaneously and reveal that binding of the allosteric effector ppGpp reshapes the RNAP-DksA complex. The structural data support a model for transcriptional inhibition in which ppGpp potentiates the destabilization of open complexes by DksA. This work establishes a structural basis for understanding the pleiotropic effects of DksA and ppGpp on transcriptional regulation in proteobacteria.

Structural basis of transcription-translation coupling

“…Movies S1-S6 Supplemental References (26-42) E. coli RNAP core enzyme E. coli RNAP core enzyme was prepared from E. coli strain BL21 Star (DE3) (ThermoFisher) transformed with plasmid pVS10 (26; encodes E. coli RNAP β' with C-terminal hexahistidine tag, β, α, and ω subunits), as described (27). The product (purity >95%) was stored in aliquots in RNAP storage buffer (10 mM Tris-HCl, pH 7.6, 100 mM NaCl, 0.1 mM EDTA, and 5 mM dithiothreitol) at -80ºC.…”

The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

“…During the stringent response, ppGpp is produced in high quantities, binds to RNAP, destabilizes RP O at ribosomal RNA (rrn) and other promoters, and inhibits isomerization of RP C to RP O at rrn and other promoters (38). ppGpp binds to two distinct sites in RNAP, "site 1" located at the interface between RNAP β' and ω subunits, and "site 2" located near the RNAP secondary-channel β' rim helices (37,(39)(40)(41). It is thought that ppGpp exerts its effects on RNAP through an allosteric mechanism, but details are unclear (37,(40)(41)(42).…”

An Introduction to the Structure and Function of the Catalytic Core Enzyme of Escherichia coli RNA Polymerase

“…The near-atomic resolution X-ray crystal structure of E. coli RNAP was determined first as holoenzyme containing σ 70 (Murakami, 2013). X-ray crystal and cryo-electron microscopy (cryo-EM) structures of E. coli RNAP are now available in several forms such as holoenzymes containing alternative σ factors (Liu et al, 2016; Yang et al, 2015), promoter DNA complexes (Glyde et al, 2017; Zuo and Steitz, 2015), elongation complex (Kang et al, 2017), in complex with transcription factors (Bae et al, 2013; Liu et al, 2017; Molodtsov et al, 2018), and with inhibitors/antibiotics (Bae et al, 2015; Chen et al, 2017; Degen et al, 2014; Molodtsov et al, 2015; Molodtsov et al, 2013), providing details of the structure and function of E. coli RNAP transcription and regulation.…”

“…Recent studies identified another ppGpp binding site near the secondary channel of RNAP, which is ~60 Å away from the site 1. The second ppGpp binding site is formed only in the presence of the RNAP binding transcription regulator DksA (Molodtsov et al, 2018; Ross et al, 2016). …”

“…4A) (Zhang et al, 2015). The secondary channel acts as a binding cavity for accessory factors that modulate RNAP activity such as Gre factors (Opalka et al, 2003) and that regulate transcription such as DksA and TraR (Molodtsov et al, 2018). Two additional motifs from the β’ subunit play important roles in the RNA synthesis reaction: the trigger loop for the catalysis and the bridge helix for the DNA/RNA translocation during the nucleotide addition cycle (Fig.…”