DNA recognition by Escherichia coli CbpA protein requires a conserved arginine–minor-groove interaction

Chintakayala, Kiran; Sellars, Laura; Singh, Shivani; Shahapure, Rajesh; Westerlaken, Ilja; Meyer, Anne S.; Dame, Remus T.; Grainger, David C.

doi:10.1093/nar/gkv012

Cited by 10 publications

(14 citation statements)

References 27 publications

(54 reference statements)

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“…Although the L p cannot be directly calculated with TPM, the change in RMS is a reliable measure for protein binding. TPM has been used to measure DNA-binding by architectural proteins, proteins involved in DNA replication, transcription, DNA repair, and recombination [12][13][14][15][16][17][18][19][20][21]. A bead is tethered to a glass surface by DNA.…”

Section: Introductionmentioning

confidence: 99%

Quantitation of DNA-Binding Affinity Using Tethered Particle Motion

Henneman¹,

Heinsman²,

Battjes³

et al. 2018

Methods in Molecular Biology

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The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered Particle Motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to reliably measure binding constants of proteins binding to DNA, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This makes it possible to measure binding properties. We use the bacterial protein Integration Host Factor (IHF) as an example to show how specific binding to DNA can be measured. Moreover, we show a new intuitive quantitative approach to displaying data obtained via TPM.

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Section: Introductionmentioning

confidence: 99%

Quantitation of DNA-Binding Affinity Using Tethered Particle Motion

Henneman¹,

Heinsman²,

Battjes³

et al. 2018

Methods in Molecular Biology

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“…In addition, we observed a lower degree of conservation in the other protein domains and especially (32% identity) in the linker-CDTI region (Figure 1). Importantly, we noticed a low degree of conservation of amino acids previously reported to be crucial for E. coli DNA-binding activity (indicated by grey and black arrowheads in Figure 1 [8,11]). Finally, of interest is the observation that of the two residues, W287 and L290, crucial for E. coli CbpA dimerization [10], only one is conserved in the H. pylori CbpA protein ( Figure 1, empty arrowheads).…”

Section: Sequence Similarity Between Helicobacter Pylori (H Pylori) mentioning

confidence: 74%

“…The experimentally characterized functional domains of E. coli CbpA are indicated as follows: J-domain (aa 1 to aa 75): green; linker (aa 76 to 117): not highlighted; CTDI (aa 118 to aa 201): cyan; CTDII (aa 202 to aa 306): red. Grey and black arrowheads indicate residues involved in DNA binding (according [8] and [11], respectively), while empty arrowheads denote crucial residues for CbpA dimerization (according to [10]).…”

Section: Sequence Similarity Between Helicobacter Pylori (H Pylori) mentioning

confidence: 99%

“…In addition, it has been demonstrated that CbpA accumulates in the stationary phase of bacterial growth and, following interaction with DNA and the formation of protein-DNA aggregates, it is able to protect nucleic acids from damage, acting as a nucleoid-associated protein [10]. The detailed mechanism of DNA binding has been dissected recently and shown to involve the recognition of the DNA minor groove [11]. Of note is the observation that both co-chaperone and DNA-binding activities of CbpA are regulated through direct interaction with a specific partner protein called CbpM (for CbpA modulator).…”

Section: Introductionmentioning

confidence: 99%

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The Helicobacter pylori HspR-Modulator CbpA Is a Multifunctional Heat-Shock Protein

2020

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The medically important human pathogen Helicobacter pylori relies on a collection of highly conserved heat-shock and chaperone proteins to preserve the integrity of cellular polypeptides and to control their homeostasis in response to external stress and changing environmental conditions. Among this set of chaperones, the CbpA protein has been shown to play a regulatory role in heat-shock gene regulation by directly interacting with the master stress-responsive repressor HspR. Apart from this regulatory role, little is known so far about CbpA functional activities. Using biochemistry and molecular biology approaches, we have started the in vitro functional characterization of H. pylori CbpA. Specifically, we show that CbpA is a multifunctional protein, being able to bind DNA and to stimulate the ATPase activity of the major chaperone DnaK. In addition, we report a preliminary observation suggesting that CbpA DNA-binding activity can be affected by the direct interaction with the heat-shock master repressor HspR, supporting the hypothesis of a reciprocal crosstalk between these two proteins. Thus, our work defines novel functions for H. pylori CbpA and stimulates further studies aimed at the comprehension of the complex regulatory interplay among chaperones and heat-shock transcriptional regulators.

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“…The involvement of an arginine residue in the shape readout of a narrow minor groove seems to be a common phenomenon for many transcription factors (Rohs et al, 2009; Rohs et al, 2010). Interestingly, this type of protein-DNA interaction occurs in prokaryotes as well as in eukaryotes and also in completely unrelated DNA-binding protein families (Rohs et al, 2009) with an increasing number of representatives being described in recent years (Mendieta et al, 2012; Quade et al, 2012; Alanazi et al, 2013; Porrua et al, 2013; Stevenson et al, 2013; Chintakayala et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

The floral homeotic protein SEPALLATA3 recognizes target DNA sequences by shape readout involving a conserved arginine residue in the MADS-domain

Gusewski

Melzer

Rümpler

et al. 2017

Preprint

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SEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor and acentral player in flower development. MADS-domain proteins bind as dimers to ATrich sequences termed 'CArG-boxes' which share the consensus 5'-CC(A/T) 6 GG-3'.Since only a fraction of the abundant CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-box sequences into genuine recognition sites.Here, we investigated to which extent the shape of the DNA contributes to the DNAbinding specificity of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to a variety of DNA probes which all contain the CArG-box motif, but differ in their DNA shape characteristics. We found that binding affinity correlates well with certain DNA shape features associated with 'A-tracts'. Analysis of SEPALLATA3 proteins with single amino acid substitutions in the DNA-binding MADS-domain further revealed that a highly conserved arginine residue, which is expected to contact the DNA minor groove, contributes significantly to the shape readout. Our studies show that the specific recognition of cis-regulatory elements by plant MADSdomain transcription factors heavily depend on shape readout mechanisms and that the absence of a critical arginine residue in the MADS-domain impairs binding specificity. peer-reviewed)

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DNA recognition by Escherichia coli CbpA protein requires a conserved arginine–minor-groove interaction

Cited by 10 publications

References 27 publications

Quantitation of DNA-Binding Affinity Using Tethered Particle Motion

Quantitation of DNA-Binding Affinity Using Tethered Particle Motion

The Helicobacter pylori HspR-Modulator CbpA Is a Multifunctional Heat-Shock Protein

The floral homeotic protein SEPALLATA3 recognizes target DNA sequences by shape readout involving a conserved arginine residue in the MADS-domain

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