Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription

Abstract

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ 70 -dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator−DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)-a Cu + -responsive MerR-family metalloregulator-modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR−PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the deadend and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo-and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.single-molecule FRET | protein-DNA interaction dynamics | MerR-family regulators | metal-responsive transcription regulation M aintaining cellular metal homeostasis is essential for bacteria, which often dwell in environments with high concentrations of metals. Some of these metals are purely toxic to bacteria, such as Cd and Hg. Many others are required for cellular function, such as Zn and Cu, but can be toxic in excess. Cells have thus developed many ways to regulate intracellular metal concentrations (1-9). Metal-responsive transcriptional regulation is one of them, where metalloregulators respond to intracellular metal ions and regulate transcription of metal efflux, uptake, or other metal homeostasis genes (4-9).In Gram-negative bacteria, MerR-family metalloregulators act on σ 70 -dependent suboptimal promoters to repress or activate transcription of metal resistance genes (7,8). These suboptimal promoters have elongated spacing, 19-20 bp (Fig. 1), compared with the optimal 17 ± 1 bp, between the −35 and −10 elements. This elongated spacing causes a misalignment of these two recognition elements, impairing proper interactions with the RNA polymerase (RNAP) and leading to a weak basal leve...

The CpxR/CpxA system contributes to Salmonella gold‐resistance by controlling the GolS‐dependent gesABC transcription

“…This metalloregulator interacts with operators located at atypical σ 70 ‐dependent promoters with a 19 bp‐long spacer between the −35 and −10 elements instead of the optimal 17 ± 1 bp. Binding of Au(I) to GolS is proposed to be essential to remodel the promoter structure aligning the −35 and −10 elements for their simultaneous recognition by the RNA polymerase (Perez Audero et al ., ; Humbert et al ., ; Martell et al ., ; Philips et al ., ). The sequence of the GolS‐operator at the gesABC promoter differs from those at golTS or golB , the other two transcriptional units of the Gol regulon (Checa et al ., ; Perez Audero et al ., ).…”

Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins

“…Many biophysical methods have been applied to characterize the proteins taking part in the copper cycle: (1) X-ray, nuclear magnetic resonance (NMR), and cryo-electron microscopy (EM) have been used to resolve the structures of various proteins [14,15,16,17,18,19,20]; (2) extended x-ray adsorption fine structure (EXAFS) was used to define the Cu(I) binding sites [21,22]; (3) single-molecule Forster resonance energy transfer (smFRET) has been employed to study the dynamics of these systems [23,24,25]; (4) electron paramagnetic resonance (EPR) measurements have been used to capture the various conformational states of these proteins [26,27,28,29]; (5) molecular simulations have been applied to fill many gaps of knowledge and to decipher unclear aspects of this intricate regulatory mechanism [30,31,32,33].…”

CopR, a global regulator of transcription to maintain copper homeostasis inPyrococcus furiosus

“…Such behaviour would be very similar to the function of the copper-sensing transcription factor CueR from Escherichia coli (64). CueR can activate transcription by controlling open complex formation, while it is continuously bound to DNA (65). This mechanism may permit more rapid responses to environmental changes.…”