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Engineered holliday junctions as single-molecule reporters for protein-DNA interactions with application to a MerR-family regulator.

ABSTRACT: Protein-DNA interactions are essential for gene maintenance, replication, and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here, we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs. Because the effects of protein binding are converted to the changes in the structure and dynamics of HJs, protein-DNA interactions that involve small structural changes of DNA can be studied. We apply this method to investigate how the MerR-family regulator PbrR691 interacts with DNA for transcriptional regulation. Both apo- and holo-PbrR691 bind the stacked conformers of the engineered HJ, change their structures, constrain their conformational distributions, alter the kinetics, and shift the equilibrium of their structural dynamics. The information obtained maps the potential energy surfaces of HJ before and after PbrR691 binding and reveals the protein actions that force DNA structural changes for transcriptional regulation. The ability of PbrR691 to bind both HJ conformers and still allow HJ structural dynamics also informs about its conformational flexibility that may have significance for its regulatory function. This method of using engineered HJs offers quantification of the changes both in structure and in dynamics of DNA upon protein binding and thus provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.

SUBMITTER: Sarkar SK

PROVIDER: S-EPMC2528078 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Engineered holliday junctions as single-molecule reporters for protein-DNA interactions with application to a MerR-family regulator.

Sarkar Susanta K SK Andoy Nesha May NM Benítez Jaime J JJ Chen Peng R PR Kong Jason S JS He Chuan C Chen Peng P

Journal of the American Chemical Society 20070920 41

Protein-DNA interactions are essential for gene maintenance, replication, and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here, we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs ...[more]

PMID: 17880214

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Project description:The MerR family of transcriptional regulators are dimeric proteins with an N-terminal helix-turn-helix DNA binding domain (DBD), followed by an antiparallel coiled-coil subunit interaction region, and usually by a C-terminal effector binding domain (EBD). This family is distinguished by the high amino acid similarity in the DBD and low similarity in the EBD, consistent with the role of this domain in each protein in sensing effector molecules. Most members of the family are activators, and act at promoters with long spacer regions and respond to the binding of inducers by distorting the promoter DNA to allow open complex formation and transcriptional activation. In MerR proteins the EBD contains a metal binding pocket formed by three cysteines. This Cys center characteristic of MerR regulators is primarily suited for metal binding and mediates activation of metal detoxification systems in bacteria. Over the last decade, it has become clear that the MerR family of regulators is more diverse than originally recognized. The availability of completed bacterial genome sequences has enabled the search for additional types of MerR-like regulators. We here present a novel MerR-like transcription factor lacking an EBD involved in iron uptake regulation (MliR) present in the marine bacterium Bizionia argentinensis (JUB59). An in silico analysis revealed that homologues of the MliR protein are widely distributed among different bacterial species. Deletion of the mliR gene led to decreased cell growth, increased cell adhesion and filamentation. RNA sequencing analysis showed that expression of several iron uptake-related mRNAs were downregulated in mliR-deletion mutant. Through NMR-based metabolomics, ICP-MS, fluorescence microscopy and biochemical analysis we evaluated metabolic and phenotypic changes associated with mliR deletion. This work provides the first evidence of a MerR-family regulator involved in iron uptake and contributes to expanding our current knowledge on iron metabolism.in bacteria.

Dataset Information

Engineered holliday junctions as single-molecule reporters for protein-DNA interactions with application to a MerR-family regulator.

Publications

Engineered holliday junctions as single-molecule reporters for protein-DNA interactions with application to a MerR-family regulator.

Similar Datasets

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