Insight Into The Mechanisms Of Transcription Regulation By A Merr-Family Metalloregulator Through Single-Molecule Analysis
Metalloregulators regulate transcription in response to metal ions. MerR-family metalloregulators act on suboptimal promoters and operate via a unique DNA distortion mechanism, where both apo and holo bind to sequences within promoters, distorting DNA and leading to transcription repression or activation, respectively. It remains unclear, however, how these metalloregulatorDNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for repression and activation. How transcription is turned off after activation also remains unclear. Using single-molecule FRET, we studied (i) dynamic interactions of the copper efflux regulator, CueR (a Cu+-responsive MerR-family metalloregulator), with DNA, (ii) how CueR modulates RNAP interactions with the suboptimal promoter PcopA, and (iii) how RNAP affects CueRPcopA interactions. Besides quantifying CueR's DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips binding orientations at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA, facilitating recognition localization. Most strikingly, a CueR coming from solution can directly substitute a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are first such examples for any DNA-binding protein. The kinetics of direct protein substitution and assisted dissociation reactions indicate that these two novel processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely. We also found that RNAP forms two non-interconverting complexes at PcopA, a terminal dead-end complex and an open complex, constituting a branched mechanistic pathway distinct from the prevalent linear pathways for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of a "dynamic equilibrium shift" mechanism in regulating transcription initiation. It modulates RNAP's binding-unbinding kinetics, either, in its apo-repressor form, reinforcing the dominance of the terminal dead-end complex to repress transcription or, in its holo-activator form, shifting the interactions toward the open complex to activate transcription, without allowing interconversion between the dead-end and open complexes. RNAP in turn locks CueR-PcopA binding into its specific binding mode, likely amplifying DNA structural changes imposed by apo- and holo-CueR, leading to a synergistic effect in forming ternary RNAP-PcopA-CueR complexes.
Single-molecule FRET; Protein–DNA interaction dynamics; Metal-mediated transcription regulation
Zipfel,Warren R.; Lin,Hening
Chemistry and Chemical Biology
Ph.D. of Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis