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Richard H. Ebright - One of the best experts on this subject based on the ideXlab platform.
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Structural Basis of Transcription Activation: The CAP-CTD-DNA Complex
2016Co-Authors: Gary Parkinson, Helen M Berman, Jinsong Liu, Erich Blatter, Yon W. Ebright, Richard H. EbrightAbstract:The Escherichia coli Catabolite Activator Protein (CAP) activates transcription at Plac, Pgal, and other promoters through interactions with the RNA polymerase subunit carboxyl-terminal domain (CTD). We determined the crystal struc-ture of the CAP-CTD-DNA complex at a resolution of 3.1 angstroms. CAP makes direct Protein-Protein interactions with CTD, and CTD makes direct Protein-DNA interactions with the DNA segment adjacent to the DNA site for CAP. There are no large-scale conformational changes in CAP and CTD, and the interface between CAP andCTD is small. These findings are consistentwith the proposal that activation involves a simple “recruitment ” mechanism. The Catabolite Activator Protein (CAP) [also referred to as the cyclic adenosine monophos-phate (cAMP) receptor Protein] activates transcription by binding to a DNA site locat-ed in or upstream of the core promoter and interacting with the RNA polymeras
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three dimensional em structure of an intact Activator dependent transcription initiation complex
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Brian P Hudson, Richard H. Ebright, Joel Quispe, Samuel Laragonzalez, Younggyu Kim, Helen M Berman, Eddy Arnold, Catherine L LawsonAbstract:Abstract We present the experimentally determined 3D structure of an intact Activator-dependent transcription initiation complex comprising the Escherichia coli Catabolite Activator Protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions −78 to +20 of a Class I CAP-dependent promoter with a CAP site at position −61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP α subunit C-terminal domain (αCTD), interactions of αCTD with σ70 region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP β′, β, and σ70 subunits.
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structural basis for camp mediated allosteric control of the Catabolite Activator Protein
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Nataliya Popovych, Richard H. Ebright, Shiouru Tzeng, Marco Tonelli, Charalampos G KalodimosAbstract:The cAMP-mediated allosteric transition in the Catabolite Activator Protein (CAP; also known as the cAMP receptor Protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory Proteins.
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dynamically driven Protein allostery
Nature Structural & Molecular Biology, 2006Co-Authors: Nataliya Popovych, Richard H. Ebright, Charalampos G KalodimosAbstract:Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the Protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in Protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric Catabolite Activator Protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching Protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.
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Catabolite Activator Protein dna binding and transcription activation
Current Opinion in Structural Biology, 2004Co-Authors: Catherine L Lawson, Richard H. Ebright, Helen M Berman, David Swigon, Katsuhiko S Murakami, Seth A DarstAbstract:Abstract Recently determined structures of the Escherichia coli Catabolite Activator Protein (CAP) in complex with DNA, and in complex with the RNA polymerase α subunit C-terminal domain (αCTD) and DNA, have yielded insights into how CAP binds DNA and activates transcription. Comparison of multiple structures of CAP–DNA complexes has revealed the contributions of direct and indirect readout to DNA binding by CAP. The structure of the CAP–αCTD–DNA complex has provided the first structural description of interactions between a transcription Activator and its functional target within the general transcription machinery. Using the structure of the CAP–αCTD–DNA complex, the structure of an RNA polymerase–DNA complex, and restraints from biophysical, biochemical and genetic experiments, it has been possible to construct detailed three-dimensional models of intact class I and class II transcription activation complexes.
Helen M Berman - One of the best experts on this subject based on the ideXlab platform.
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Structural Basis of Transcription Activation: The CAP-CTD-DNA Complex
2016Co-Authors: Gary Parkinson, Helen M Berman, Jinsong Liu, Erich Blatter, Yon W. Ebright, Richard H. EbrightAbstract:The Escherichia coli Catabolite Activator Protein (CAP) activates transcription at Plac, Pgal, and other promoters through interactions with the RNA polymerase subunit carboxyl-terminal domain (CTD). We determined the crystal struc-ture of the CAP-CTD-DNA complex at a resolution of 3.1 angstroms. CAP makes direct Protein-Protein interactions with CTD, and CTD makes direct Protein-DNA interactions with the DNA segment adjacent to the DNA site for CAP. There are no large-scale conformational changes in CAP and CTD, and the interface between CAP andCTD is small. These findings are consistentwith the proposal that activation involves a simple “recruitment ” mechanism. The Catabolite Activator Protein (CAP) [also referred to as the cyclic adenosine monophos-phate (cAMP) receptor Protein] activates transcription by binding to a DNA site locat-ed in or upstream of the core promoter and interacting with the RNA polymeras
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three dimensional em structure of an intact Activator dependent transcription initiation complex
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Brian P Hudson, Richard H. Ebright, Joel Quispe, Samuel Laragonzalez, Younggyu Kim, Helen M Berman, Eddy Arnold, Catherine L LawsonAbstract:Abstract We present the experimentally determined 3D structure of an intact Activator-dependent transcription initiation complex comprising the Escherichia coli Catabolite Activator Protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions −78 to +20 of a Class I CAP-dependent promoter with a CAP site at position −61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP α subunit C-terminal domain (αCTD), interactions of αCTD with σ70 region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP β′, β, and σ70 subunits.
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Catabolite Activator Protein dna binding and transcription activation
Current Opinion in Structural Biology, 2004Co-Authors: Catherine L Lawson, Richard H. Ebright, Helen M Berman, David Swigon, Katsuhiko S Murakami, Seth A DarstAbstract:Abstract Recently determined structures of the Escherichia coli Catabolite Activator Protein (CAP) in complex with DNA, and in complex with the RNA polymerase α subunit C-terminal domain (αCTD) and DNA, have yielded insights into how CAP binds DNA and activates transcription. Comparison of multiple structures of CAP–DNA complexes has revealed the contributions of direct and indirect readout to DNA binding by CAP. The structure of the CAP–αCTD–DNA complex has provided the first structural description of interactions between a transcription Activator and its functional target within the general transcription machinery. Using the structure of the CAP–αCTD–DNA complex, the structure of an RNA polymerase–DNA complex, and restraints from biophysical, biochemical and genetic experiments, it has been possible to construct detailed three-dimensional models of intact class I and class II transcription activation complexes.
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indirect readout of dna sequence at the primary kink site in the cap dna complex alteration of dna binding specificity through alteration of dna kinking
Journal of Molecular Biology, 2001Co-Authors: Shengfeng Chen, Richard H. Ebright, Angelo Gunasekera, Xiaoping Zhang, Thomas A Kunkel, Helen M BermanAbstract:Abstract The Catabolite Activator Protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of ∼40° and a twist angle of ∼20°, between positions 6 and 7 of the DNA half-site, 5′-A 1 A 2 A 3 T 4 G 5 T 6 G 7 A 8 T 9 C 10 T 11 -3′ (“primary kink”). CAP recognizes the base-pair immediately 5′ to the primary-kink site, T:A 6 , through an “indirect-readout” mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3′ to the primary-kink site, G:C 7 , through a “direct-readout” mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C 7 . Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A 6 to specificity for C:G 6 , and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181→Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A 6 . The Glu181→Asp substitution does not eliminate hydrogen-bond formation with G:C 7 , and thus does not eliminate direct-readout-based specificity for G:C 7 .
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indirect readout of dna sequence at the primary kink site in the cap dna complex dna binding specificity based on energetics of dna kinking
Journal of Molecular Biology, 2001Co-Authors: Shengfeng Chen, Richard H. Ebright, Jaroslav Vojtechovsky, Gary N Parkinson, Helen M BermanAbstract:The Catabolite Activator Protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is Protein-DNA interaction, and not DNA sequence.
Robert A. Bender - One of the best experts on this subject based on the ideXlab platform.
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alanine catabolism in klebsiella aerogenes molecular characterization of the dadab operon and its regulation by the nitrogen assimilation control Protein
Journal of Bacteriology, 1998Co-Authors: Brian K Janes, Robert A. BenderAbstract:Klebsiella aerogenes strains with reduced levels of d-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control Protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position −44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the Catabolite Activator Protein and the leucine-responsive regulatory Protein.
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alanine catabolism in klebsiella aerogenes molecular characterization of the dadab operon and its regulation by the nitrogen assimilation control Protein
Journal of Bacteriology, 1998Co-Authors: Brian K Janes, Robert A. BenderAbstract:Klebsiella aerogenes strains with reduced levels of d-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control Protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position −44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the Catabolite Activator Protein and the leucine-responsive regulatory Protein.
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roles of Catabolite Activator Protein sites centered at 81 5 and 41 5 in the activation of the klebsiella aerogenes histidine utilization operon hutuh
Journal of Bacteriology, 1994Co-Authors: Robert Osuna, Brian K Janes, Robert A. BenderAbstract:The Klebsiella aerogenes hutUH operon is preceded by a promoter region, hut(P), that contains two divergent promoters (hutUp and Pc) which overlap and are alternately expressed. In the absence of the Catabolite gene Activator Protein-cyclic AMP (CAP-cAMP) complex, Pc is predominantly expressed while hutUp is largely repressed. CAP-cAMP has the dual effect of repressing transcription from Pc while simultaneously activating transcription from hutUp. DNA deletion mutations in this region were used to identify DNA sequences required for transcription of these two promoters. We showed that inactivation of Pc by DNA deletion did not result in activation of hutUp in vitro or in vivo. In addition, Escherichia coli CAP mutants that are known to bind and bend DNA normally but are unable to activate various CAP-dependent promoters were also unable to activate hutUp in vivo. These results invalidate an indirect activation model by which CAP-mediated repression of Pc in itself would led to activation of hutUp. Gel retardation asays with various deletion mutations of hut(P) and DNase I protection analyses revealed a high-affinity CAP binding site (CAP site 1) centered at -81.5 relative to the hutUp start of transcription and a second low-affinity CAP site (CAP site 2) centered at about -41.5. CAP site 1 is essential for activation of hutUp. Although CAP site 2 by itself is unable to activate hutUp in vivo under Catabolite-activating conditions, it appears to be required for maximal transcription from a site centered at -41.5, does not activate hutUp suggests that the role of CAP-cAMP at the weaker CAP site may be different from that of other promoters containing a similarly positioned site. We propose that CAP directly stimulates the activity of RNA polymerase at hutUp and that this reaction is completely dependent on a naturally occurring CAP site centered at -81.5 and also involves a second CAP site centered at about -41.5 for maximal activation.
Charalampos G Kalodimos - One of the best experts on this subject based on the ideXlab platform.
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dynamic activation of an allosteric regulatory Protein
Nature, 2009Co-Authors: Shiouru Tzeng, Charalampos G KalodimosAbstract:Effector molecules are thought to control the activity of allosteric Proteins by binding to an allosteric site, distinct from the active site, thereby inducing and stabilizing a specific conformational state of the Protein. A new study suggests that the notion of purely structurally regulated activity in allosteric Proteins should be revised to include a frequently dominating contribution from Protein dynamics. Shiou-Ru Tzeng and Charalampos Kalodimos characterized cyclic AMP binding to Catabolite Activator Protein (CAP), a transcriptional Activator often used as a model for allostery. They find, surprisingly, that even when in a structurally inactive conformation, CAP can be activated for ligand (DNA) binding by changes in Protein dynamics. Allosteric regulation is used to control Protein activity across a broad range of biological processes. Effectors are thought to function by selectively stabilizing a specific conformational state with distinct binding or enzymatic activity, thereby regulating Protein activity. Here, the characterization of the binding of cyclic AMP to the Catabolite Activator Protein demonstrates that allosteric Proteins can be regulated predominantly by changes in their structural dynamics. Allosteric regulation is used as a very efficient mechanism to control Protein activity in most biological processes, including signal transduction, metabolism, catalysis and gene regulation1,2,3,4,5,6. Allosteric Proteins can exist in several conformational states with distinct binding or enzymatic activity. Effectors are considered to function in a purely structural manner by selectively stabilizing a specific conformational state, thereby regulating Protein activity. Here we show that allosteric Proteins can be regulated predominantly by changes in their structural dynamics. We have used NMR spectroscopy and isothermal titration calorimetry to characterize cyclic AMP (cAMP) binding to the Catabolite Activator Protein (CAP), a transcriptional Activator that has been a prototype for understanding effector-mediated allosteric control of Protein activity7. cAMP switches CAP from the ‘off’ state (inactive), which binds DNA weakly and non-specifically, to the ‘on’ state (active), which binds DNA strongly and specifically. In contrast, cAMP binding to a single CAP mutant, CAP-S62F, fails to elicit the active conformation; yet, cAMP binding to CAP-S62F strongly activates the Protein for DNA binding. NMR and thermodynamic analyses show that despite the fact that CAP-S62F-cAMP2 adopts the inactive conformation, its strong binding to DNA is driven by a large conformational entropy originating in enhanced Protein motions induced by DNA binding. The results provide strong evidence that changes in Protein motions may activate allosteric Proteins that are otherwise structurally inactive.
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structural basis for camp mediated allosteric control of the Catabolite Activator Protein
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Nataliya Popovych, Richard H. Ebright, Shiouru Tzeng, Marco Tonelli, Charalampos G KalodimosAbstract:The cAMP-mediated allosteric transition in the Catabolite Activator Protein (CAP; also known as the cAMP receptor Protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory Proteins.
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dynamically driven Protein allostery
Nature Structural & Molecular Biology, 2006Co-Authors: Nataliya Popovych, Richard H. Ebright, Charalampos G KalodimosAbstract:Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the Protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in Protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric Catabolite Activator Protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching Protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.
Shengfeng Chen - One of the best experts on this subject based on the ideXlab platform.
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indirect readout of dna sequence at the primary kink site in the cap dna complex alteration of dna binding specificity through alteration of dna kinking
Journal of Molecular Biology, 2001Co-Authors: Shengfeng Chen, Richard H. Ebright, Angelo Gunasekera, Xiaoping Zhang, Thomas A Kunkel, Helen M BermanAbstract:Abstract The Catabolite Activator Protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of ∼40° and a twist angle of ∼20°, between positions 6 and 7 of the DNA half-site, 5′-A 1 A 2 A 3 T 4 G 5 T 6 G 7 A 8 T 9 C 10 T 11 -3′ (“primary kink”). CAP recognizes the base-pair immediately 5′ to the primary-kink site, T:A 6 , through an “indirect-readout” mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3′ to the primary-kink site, G:C 7 , through a “direct-readout” mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C 7 . Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A 6 to specificity for C:G 6 , and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181→Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A 6 . The Glu181→Asp substitution does not eliminate hydrogen-bond formation with G:C 7 , and thus does not eliminate direct-readout-based specificity for G:C 7 .
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indirect readout of dna sequence at the primary kink site in the cap dna complex dna binding specificity based on energetics of dna kinking
Journal of Molecular Biology, 2001Co-Authors: Shengfeng Chen, Richard H. Ebright, Jaroslav Vojtechovsky, Gary N Parkinson, Helen M BermanAbstract:The Catabolite Activator Protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is Protein-DNA interaction, and not DNA sequence.