Investigation of DNA sequence recognition by a streptomycete MarR family transcriptional regulator through surface plasmon resonance and X-ray crystallography

TY - JOUR

T1 - Investigation of DNA sequence recognition by a streptomycete MarR family transcriptional regulator through surface plasmon resonance and X-ray crystallography

AU - Stevenson, Clare E M

AU - Assaad, Aoun

AU - Chandra, Govind

AU - Le, Tung B K

AU - Greive, Sandra J

AU - Bibb, Mervyn J

AU - Lawson, David M

PY - 2013/8

Y1 - 2013/8

N2 - Consistent with their complex lifestyles and rich secondary metabolite profiles, the genomes of streptomycetes encode a plethora of transcription factors, the vast majority of which are uncharacterized. Herein, we use Surface Plasmon Resonance (SPR) to identify and delineate putative operator sites for SCO3205, a MarR family transcriptional regulator from Streptomyces coelicolor that is well represented in sequenced actinomycete genomes. In particular, we use a novel SPR footprinting approach that exploits indirect ligand capture to vastly extend the lifetime of a standard streptavidin SPR chip. We define two operator sites upstream of sco3205 and a pseudopalindromic consensus sequence derived from these enables further potential operator sites to be identified in the S. coelicolor genome. We evaluate each of these through SPR and test the importance of the conserved bases within the consensus sequence. Informed by these results, we determine the crystal structure of a SCO3205-DNA complex at 2.8 Å resolution, enabling molecular level rationalization of the SPR data. Taken together, our observations support a DNA recognition mechanism involving both direct and indirect sequence readout.

AB - Consistent with their complex lifestyles and rich secondary metabolite profiles, the genomes of streptomycetes encode a plethora of transcription factors, the vast majority of which are uncharacterized. Herein, we use Surface Plasmon Resonance (SPR) to identify and delineate putative operator sites for SCO3205, a MarR family transcriptional regulator from Streptomyces coelicolor that is well represented in sequenced actinomycete genomes. In particular, we use a novel SPR footprinting approach that exploits indirect ligand capture to vastly extend the lifetime of a standard streptavidin SPR chip. We define two operator sites upstream of sco3205 and a pseudopalindromic consensus sequence derived from these enables further potential operator sites to be identified in the S. coelicolor genome. We evaluate each of these through SPR and test the importance of the conserved bases within the consensus sequence. Informed by these results, we determine the crystal structure of a SCO3205-DNA complex at 2.8 Å resolution, enabling molecular level rationalization of the SPR data. Taken together, our observations support a DNA recognition mechanism involving both direct and indirect sequence readout.

KW - Bacterial Proteins

KW - Crystallography, X-Ray

KW - DNA, Bacterial

KW - DNA, Intergenic

KW - Models, Molecular

KW - Operator Regions, Genetic

KW - Protein Binding

KW - Protein Footprinting

KW - Streptomyces coelicolor

KW - Surface Plasmon Resonance

KW - Transcription Factors

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1093/nar/gkt523

DO - 10.1093/nar/gkt523

M3 - Article

C2 - 23748564

SN - 0305-1048

VL - 41

SP - 7009

EP - 7022

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 14

ER -