Affinity, Specificity, and Cooperativity of DNA Binding by Bacterial Gene Regulatory Proteins

Nearly all of biology depends on interactions between molecules: proteins with small molecules, proteins with other proteins, nucleic acids with small molecules, and nucleic acids with proteins that regulate gene expression, our concern in this Special Issue. All those kinds of interactions, and others, constitute the vast majority of biology at the molecular level. An understanding of those interactions requires that we quantify them to learn how they interact: How strongly? With which partners? How—and how well—are different partners distinguished? This review addresses the evolution of our current understanding of the molecular origins of affinity and specificity in regulatory protein–DNA interactions, and suggests that both these properties can be modulated by cooperativity.

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Mutations in Transcriptional Regulators Allow Selective Engineering of Signal Integration Logic

mBio ◽

10.1128/mbio.01171-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 4

Author(s):

Szabolcs Semsey

Keyword(s):

Gene Regulatory Networks ◽

Regulatory Networks ◽

Regulatory Protein ◽

Point Mutations ◽

Building Blocks ◽

Regulatory Elements ◽

Regulatory Proteins ◽

Single Amino Acid ◽

Signal Integration ◽

Gene Regulatory

ABSTRACT Bacterial cells monitor their environment by sensing a set of signals. Typically, these environmental signals affect promoter activities by altering the activity of transcription regulatory proteins. Promoters are often regulated by more than one regulatory protein, and in these cases the relevant signals are integrated by certain logic. In this work, we study how single amino acid substitutions in a regulatory protein (GalR) affect transcriptional regulation and signal integration logic at a set of engineered promoters. Our results suggest that point mutations in regulatory genes allow independent evolution of regulatory logic at different promoters. IMPORTANCE Gene regulatory networks are built from simple building blocks, such as promoters, transcription regulatory proteins, and their binding sites on DNA. Many promoters are regulated by more than one regulatory input. In these cases, the inputs are integrated and allow transcription only in certain combinations of input signals. Gene regulatory networks can be easily rewired, because the function of cis-regulatory elements and promoters can be altered by point mutations. In this work, we tested how point mutations in transcription regulatory proteins can affect signal integration logic. We found that such mutations allow context-dependent engineering of signal integration logic at promoters, further contributing to the plasticity of gene regulatory networks.

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Shining a Spotlight on DNA: Single-Molecule Methods to Visualise DNA

Molecules ◽

10.3390/molecules24030491 ◽

2019 ◽

Vol 24 (3) ◽

pp. 491 ◽

Cited By ~ 8

Author(s):

Gurleen Kaur ◽

Jacob Lewis ◽

Antoine van Oijen

Keyword(s):

Mechanical Properties ◽

Nucleic Acids ◽

Small Molecules ◽

Single Molecule ◽

Single Molecules ◽

Dna Molecules ◽

Dna Interactions ◽

Biochemical Reactions ◽

Imaging Methods ◽

Protein Dna Interactions

The ability to watch single molecules of DNA has revolutionised how we study biological transactions concerning nucleic acids. Many strategies have been developed to manipulate DNA molecules to investigate mechanical properties, dynamics and protein–DNA interactions. Imaging methods using small molecules and protein-based probes to visualise DNA have propelled our understanding of complex biochemical reactions involving DNA. This review focuses on summarising some of the methodological developments made to visualise individual DNA molecules and discusses how these probes have been used in single-molecule biophysical assays.

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Adaptation and compensation in a bacterial gene regulatory network evolving under antibiotic selection

eLife ◽

10.7554/elife.70931 ◽

2021 ◽

Vol 10 ◽

Author(s):

Vishwa Patel ◽

Nishad Matange

Keyword(s):

Gene Regulatory Networks ◽

Regulatory Networks ◽

High Pressures ◽

Regulatory Protein ◽

Bacterial Gene ◽

Antibiotic Selection ◽

E Coli ◽

Evolutionary Response ◽

Evolutionary Branch ◽

Gene Regulatory

Gene regulatory networks allow organisms to generate coordinated responses to environmental challenges. In bacteria, regulatory networks are re-wired and re-purposed during evolution, though the relationship between selection pressures and evolutionary change is poorly understood. In this study, we discover that the early evolutionary response of Escherichia coli to the antibiotic trimethoprim involves derepression of PhoPQ signaling, an Mg2+-sensitive two-component system, by inactivation of the MgrB feedback-regulatory protein. We report that derepression of PhoPQ confers trimethoprim-tolerance to E. coli by hitherto unrecognized transcriptional upregulation of dihydrofolate reductase (DHFR), target of trimethoprim. As a result, mutations in mgrB precede and facilitate the evolution of drug resistance. Using laboratory evolution, genome sequencing, and mutation re-construction, we show that populations of E. coli challenged with trimethoprim are faced with the evolutionary ‘choice’ of transitioning from tolerant to resistant by mutations in DHFR, or compensating for the fitness costs of PhoPQ derepression by inactivating the RpoS sigma factor, itself a PhoPQ-target. Outcomes at this evolutionary branch-point are determined by the strength of antibiotic selection, such that high pressures favor resistance, while low pressures favor cost compensation. Our results relate evolutionary changes in bacterial gene regulatory networks to strength of selection and provide mechanistic evidence to substantiate this link.

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Bacterial Gene Regulatory Proteins: Organisation and Mechanism of Action

Molecular Microbiology ◽

10.1007/978-3-642-72071-0_7 ◽

1998 ◽

pp. 123-140 ◽

Cited By ~ 1

Author(s):

Georgina Lloyd ◽

Tamara Belyaeva ◽

Virgil Rhodius ◽

Nigel Savery ◽

Stephen Busby

Keyword(s):

Mechanism Of Action ◽

Regulatory Proteins ◽

Bacterial Gene ◽

Gene Regulatory

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Adaptation and Compensation in a Bacterial Gene Regulatory Network Evolving Under Antibiotic Selection

10.1101/2021.08.31.458319 ◽

2021 ◽

Author(s):

Vishwa Patel ◽

Nishad Matange

Keyword(s):

Gene Regulatory Networks ◽

Regulatory Networks ◽

High Pressures ◽

Regulatory Protein ◽

Bacterial Gene ◽

Antibiotic Selection ◽

E Coli ◽

Evolutionary Response ◽

Evolutionary Branch ◽

Gene Regulatory

Gene regulatory networks allow organisms to generate coordinated responses to environmental challenges. In bacteria, regulatory networks are re-wired and re-purposed during evolution, though the relationship between selection pressures and evolutionary change is poorly understood. In this study, we discover that early evolutionary response of Escherichia coli to the antibiotic trimethoprim involves de-repression of PhoPQ signalling, a Mg2+-sensitive two-component system, by inactivation of the MgrB feedback-regulatory protein. We report that de-repression of PhoPQ confers trimethoprim-tolerance to E. coli by hitherto unrecognized transcriptional up-regulation of dihydrofolate reductase (DHFR), target of trimethoprim. As a result, mutations in mgrB precede and facilitate the evolution of drug resistance. Using laboratory evolution, genome sequencing and mutation re-construction, we show that populations of E. coli challenged with trimethoprim are faced with the evolutionary "choice" of transitioning from tolerant to resistant by mutations in DHFR, or compensating for the fitness costs of PhoPQ de-repression by inactivating the RpoS sigma factor, itself a PhoPQ-target. Outcomes at this evolutionary branch-point are determined by strength of antibiotic selection, such that high pressures favour resistance, while low pressures favour cost-compensation. Our results relate evolutionary changes in bacterial gene regulatory networks to strength of selection and provide mechanistic evidence to substantiate this link.

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Structural Insights into the Recognition of DNA Defects by Small Molecules

Dalton Transactions ◽

10.1039/d0dt04289g ◽

2021 ◽

Author(s):

David Dayanidhi Paul Elisa Sundar ◽

Vaidyanathan Ganesan

Keyword(s):

Nucleic Acids ◽

Metal Complexes ◽

Small Molecules ◽

Chemical Properties ◽

Biological Applications ◽

Binding Interaction ◽

Structural Insights

Studies on the binding interaction of small molecules and nucleic acids have been explored for their biological applications. With excellent photophysical/chemical properties, numerous metal complexes have been studied as structural...

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Nucleic Acids Chemistry and Engineering: Special Issue on Nucleic Acid Conjugates for Biotechnological Applications

Applied Sciences ◽

10.3390/app11083594 ◽

2021 ◽

Vol 11 (8) ◽

pp. 3594

Author(s):

Tamaki Endoh ◽

Eriks Rozners ◽

Takashi Ohtsuki

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Genetic Information ◽

Biotechnological Applications ◽

Biological Functions ◽

Primary Sequence ◽

Special Issue

Nucleic acids not only store genetic information in their primary sequence but also exhibit biological functions through the formation of their unique structures [...]

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Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector

Gene ◽

10.1016/0378-1119(89)90459-9 ◽

1989 ◽

Vol 85 (1) ◽

pp. 15-23 ◽

Cited By ~ 207

Author(s):

Jin Kim ◽

Christian Zwieb ◽

Carl Wu ◽

Sankar Adhya

Keyword(s):

Dna Bending ◽

Regulatory Proteins ◽

Gene Regulatory

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Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle

Biochemical Journal ◽

10.1042/bj1860089 ◽

1980 ◽

Vol 186 (1) ◽

pp. 89-98 ◽

Cited By ~ 51

Author(s):

T P Walsh ◽

D J Winzor ◽

F M Clarke ◽

C J Masters ◽

D J Morton

Keyword(s):

Skeletal Muscle ◽

Binding Constant ◽

Moving Boundary ◽

Regulatory Protein ◽

Regulatory Proteins ◽

Affinity Constant ◽

Quantitative Studies ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Troponin Complex

The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 × 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 × 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant.

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