Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator

Abstract

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu þ -responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.single-molecule imaging | protein-DNA interaction dynamics B acteria often dwell in environments with high concentrations of metals. Some of these metals are essential, but many are toxic. Even the essential metals, for example iron and copper, can become detrimental above a certain concentration inside cells. Many biological processes are thus present to regulate and maintain intracellular metal homeostasis (1-9). One of them is through metalloregulators, which respond to metal ions and regulate the transcription of genes that protect the bacteria from metal-induced stress (5-7, 10). The MerR-family metalloregulators respond to many metal ions with high selectivity and sensitivity, such as Hg 2þ and Cu 2þ (5,11,12).All MerR-family metalloregulators are homodimeric proteins. They regulate transcription via a DNA distortion mechanism (5, 13-16). They recognize specific dyad-symmetric DNA sequences within a promoter, and both their apo and holo forms bind DNA tightly. In the absence of metal, the metalloregulator bends the DNA; in this configuration RNA polymerase (RNAp) cannot interact with both −10 and −35 sequences properly and transcription is repressed. Upon binding metal, the metalloregulator changes its conformation and further unwinds the DNA slightly to allow proper RNAp interactions with the −10 and −35 sequences; transcription is then activated.Although the mechanisms of transcription activation by MerRfamily metalloregulators are well-studied (5, 13-16), little is yet known about how transcription activation is reversed. Turning off transcription promptly is important, however, as the cells would not want to conti...

Enterococcal Rgg-Like Regulator ElrR Activates Expression of the elrA Operon

“…The two predicted boxes for this promoter, Ϫ35 (TTGAAC) and Ϫ10 (TATAAT), are separated by 21 bp, a fairly long element (66). Interestingly, regulators of the MerR family bind between the Ϫ35 and Ϫ10 boxes separated by long spacers and induce DNA distortion, activating transcription (67,68). It is conceivable that ElrR may act in a similar manner.…”

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

“…5C) (28). The assisted dissociation or direct substitution process of CueR on PcopA is reflected by that 〈τ bound 〉 −1 , the apparent singlemolecule rate for CueR−PcopA complex dissociation, increases with the CueR concentration in the solution; and these two processes only occur in the specific CueR−PcopA binding mode, as we have shown (28).…”

“…S12A), and the corresponding peak is buried by the large E 0 peak. The two different binding orientations of CueR Cy5 on PcopA Cy3@−41 results from that the single Cy5 label on one monomer breaks the symmetry of the homodimeric CueR, as we have shown (28). The presence of the two different CueR binding modes in each orientation cannot be resolved by their E FRET , and instead, is reflected by the distribution of the dwell time τ bound on the CueRbound E CueR-P state: it is a double-exponential distribution (Fig.…”

“…The presence of the two different CueR binding modes in each orientation cannot be resolved by their E FRET , and instead, is reflected by the distribution of the dwell time τ bound on the CueRbound E CueR-P state: it is a double-exponential distribution (Fig. 5C) (28). The assisted dissociation or direct substitution process of CueR on PcopA is reflected by that 〈τ bound 〉 −1 , the apparent singlemolecule rate for CueR−PcopA complex dissociation, increases with the CueR concentration in the solution; and these two processes only occur in the specific CueR−PcopA binding mode, as we have shown (28).…”

“…We have previously shown that singly labeled CueR (at C129; SI Appendix, section 1b) is functional, and we characterized CueR interactions with the PcopA operator site (28). We found that CueR, in both its apo and holo forms, can bind to the operator site with two different binding modes: one in which CueR recognizes the specific sequence and distorts the DNA structure, and the other in which CueR likely interacts with the DNA in a nonspecific fashion (SI Appendix, Fig.…”