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Gunter Theisen - One of the best experts on this subject based on the ideXlab platform.

  • dna binding properties of the mads domain transcription factor sepallata3 and mutant variants characterized by selex seq
    Plant Molecular Biology, 2021
    Co-Authors: Sandra Kappel, Ralf Eggeling, Florian Rumpler, Marco Groth, Rainer Melzer, Gunter Theisen
    Abstract:

    We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved Arginine Residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved Arginine Residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

  • the floral homeotic protein sepallata3 recognizes target dna sequences by shape readout involving a conserved Arginine Residue in the mads domain
    Plant Journal, 2018
    Co-Authors: Sandra Kappel, Rainer Melzer, Florian Ruempler, Christian Gafert, Gunter Theisen
    Abstract:

    SEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor (TF) and a key regulator of flower development. MADS-domain proteins bind to sequences termed 'CArG-boxes' [consensus 5'-CC(A/T)6 GG-3']. Because only a fraction of the CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-boxes into genuine recognition sites. Here, we investigate to what extent the shape of the DNA is involved in a 'shape readout' that contributes to the binding of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to DNA probes that all contain the CArG-box motif, but differ in their predicted DNA shape. We found that binding affinity correlates well with a narrow minor groove of the DNA. Substitution of canonical bases with non-standard bases supports the hypothesis of minor groove shape readout by SEPALLATA3. Analysis of mutant SEPALLATA3 proteins 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 a plant MADS-domain TF, and by inference probably also of other TFs of this type, heavily depends on shape readout mechanisms.

Masaki Otagiri - One of the best experts on this subject based on the ideXlab platform.

  • protective effects of suprofen and its methyl ester against inactivation of rabbit kidney carbonyl reductase by phenylglyoxal
    Journal of Enzyme Inhibition, 2001
    Co-Authors: Yorishige Imamura, Toshiyuki Higuchi, Masaki Otagiri
    Abstract:

    Suprofen (SF) was little reduced by rabbit kidney carbonyl reductase, whereas its methyl ester (SPM) was an efficient substrate of the enzyme. To account for the differential catalytic activities for SF and SPM, the protective effects of these compounds against the inactivation of the enzyme by phenylglyoxal (PGO) were compared. Since the carboxyl group of SP is negatively charged and one essential Arginine Residue is known to be located in the NADPH-binding site of the enzyme, the protection of SP against the inactivation of the enzyme by PGO is expected to be more effective than that of SPM lacking a carboxyl group. However, the protective effects of SP and SPM were very similar. These results suggest that in spite of evidence for the binding of SP to the coenzyme-binding site, the carboxyl group of SP fails to interact with one essential Arginine Residue located in the site.

  • chemical modification of Arginine and lysine Residues in coenzyme binding domain of carbonyl reductase from rabbit kidney indomethacin affords a significant protection against inactivation of the enzyme by phenylglyoxal
    Biochimica et Biophysica Acta, 1994
    Co-Authors: Toshiyuki Higuchi, Yorishige Imamura, Masaki Otagiri
    Abstract:

    Abstract Carbonyl reductase from rabbit kidney was inactivated by phenylglyoxal (PGO) and 2,4,6-trinitrobenzenesulfonate sodium (TNBS). NADP + protected the enzyme from the inactivations by PGO and TNBS, suggesting that essential Arginine and lysine Residues are located in coenzyme-binding domain of the enzyme. Judging from the effects of PGO-treated enzymes in the presence and in the absence of NADP + on the fluorescence intensity of NADPH, one essential Arginine Residue in coenzyme-binding domain was found to have a role in the binding of NADPH to the enzyme. Indomethacin afforded a significant protection against inactivation of the enzyme by PGO, whereas it could not protect the enzyme from the inactivation by TNBS. It is reasonable to postulate that indomethacin interacts at least in part with or near one essential Arginine Residue in coenzyme-binding domain of carbonyl reductase from rabbit kidney.

Rainer Melzer - One of the best experts on this subject based on the ideXlab platform.

  • dna binding properties of the mads domain transcription factor sepallata3 and mutant variants characterized by selex seq
    Plant Molecular Biology, 2021
    Co-Authors: Sandra Kappel, Ralf Eggeling, Florian Rumpler, Marco Groth, Rainer Melzer, Gunter Theisen
    Abstract:

    We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved Arginine Residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved Arginine Residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

  • the floral homeotic protein sepallata3 recognizes target dna sequences by shape readout involving a conserved Arginine Residue in the mads domain
    Plant Journal, 2018
    Co-Authors: Sandra Kappel, Rainer Melzer, Florian Ruempler, Christian Gafert, Gunter Theisen
    Abstract:

    SEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor (TF) and a key regulator of flower development. MADS-domain proteins bind to sequences termed 'CArG-boxes' [consensus 5'-CC(A/T)6 GG-3']. Because only a fraction of the CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-boxes into genuine recognition sites. Here, we investigate to what extent the shape of the DNA is involved in a 'shape readout' that contributes to the binding of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to DNA probes that all contain the CArG-box motif, but differ in their predicted DNA shape. We found that binding affinity correlates well with a narrow minor groove of the DNA. Substitution of canonical bases with non-standard bases supports the hypothesis of minor groove shape readout by SEPALLATA3. Analysis of mutant SEPALLATA3 proteins 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 a plant MADS-domain TF, and by inference probably also of other TFs of this type, heavily depends on shape readout mechanisms.

  • the floral homeotic protein sepallata3 recognizes target dna sequences by shape readout involving a conserved Arginine Residue in the mads domain
    bioRxiv, 2017
    Co-Authors: Sandra Gusewski, Rainer Melzer, Florian Ruempler, Christian Gafert, Guenter Theissen
    Abstract:

    SEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor and a central player in flower development. MADS-domain proteins bind as dimers to AT-rich 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 DNA-binding 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 MADS-domain transcription factors heavily depend on shape readout mechanisms and that the absence of a critical Arginine Residue in the MADS-domain impairs binding specificity.

Sandra Kappel - One of the best experts on this subject based on the ideXlab platform.

  • dna binding properties of the mads domain transcription factor sepallata3 and mutant variants characterized by selex seq
    Plant Molecular Biology, 2021
    Co-Authors: Sandra Kappel, Ralf Eggeling, Florian Rumpler, Marco Groth, Rainer Melzer, Gunter Theisen
    Abstract:

    We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved Arginine Residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved Arginine Residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

  • the floral homeotic protein sepallata3 recognizes target dna sequences by shape readout involving a conserved Arginine Residue in the mads domain
    Plant Journal, 2018
    Co-Authors: Sandra Kappel, Rainer Melzer, Florian Ruempler, Christian Gafert, Gunter Theisen
    Abstract:

    SEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor (TF) and a key regulator of flower development. MADS-domain proteins bind to sequences termed 'CArG-boxes' [consensus 5'-CC(A/T)6 GG-3']. Because only a fraction of the CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-boxes into genuine recognition sites. Here, we investigate to what extent the shape of the DNA is involved in a 'shape readout' that contributes to the binding of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to DNA probes that all contain the CArG-box motif, but differ in their predicted DNA shape. We found that binding affinity correlates well with a narrow minor groove of the DNA. Substitution of canonical bases with non-standard bases supports the hypothesis of minor groove shape readout by SEPALLATA3. Analysis of mutant SEPALLATA3 proteins 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 a plant MADS-domain TF, and by inference probably also of other TFs of this type, heavily depends on shape readout mechanisms.

Antony Parker - One of the best experts on this subject based on the ideXlab platform.

  • a single Arginine Residue is required for the interaction of the electron transferring flavoprotein etf with three of its dehydrogenase partners
    Molecular and Cellular Biochemistry, 2003
    Co-Authors: Antony Parker
    Abstract:

    The interaction of several dehydrogenases with the electron transferring flavoprotein (ETF) is a crucial step required for the successful transfer of electrons into the electron transport chain. The exact determinants regarding the interaction of ETF with its dehydrogenase partners are still unknown. Chemical modification of ETF with Arginine-specific reagents resulted in the loss, to varying degrees, of activity with medium chain acyl-coenzyme A dehydrogenase (MCAD). The kinetic profiles showed the inactivations followed pseudo-first-order kinetics for all reagents used. For activity with MCAD, maximum inactivation of ETF was accomplished by 2,3-butanedione (4% residual activity after 120 min) and it was shown that modification of one Arginine Residue was responsible for the inactivation. Almost 100% restoration of this ETF activity was achieved upon incubation with free Arginine. However, the same 2,3-butanedione modified ETF only possessed decreased activity with dimethylglycine- (DMGDH, 44%) and sarcosine- (SDH, 27%) dehydrogenases unlike the abolition with MCAD. Full protection of ETF from Arginine modification by 2,3-butanedione was achieved using substrate-protected DMGDH, MCAD and SDH respectively. Cross-protection studies of ETF with the three dehydrogenases implied use of the same single Arginine Residue in the binding of all three dehydrogenases. These results lead us to conclude that this single Arginine Residue is essential in the binding of the ETF to MCAD, but only contributes partially to the binding of ETF to SDH and DMGDH and thus, the determinants of the dehydrogenase binding sites overlap but are not identical.