scholarly journals Influence of association state and DNA binding on the O2-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator

2014 ◽  
Vol 463 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Jason C. Crack ◽  
Melanie R. Stapleton ◽  
Jeffrey Green ◽  
Andrew J. Thomson ◽  
Nick E. Le Brun
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Reaction Kinetics ◽  
Nitrate Reduction ◽  
Amino Acid Substitutions ◽  
Association State

DNA-binding of [4Fe-4S] FNR and amino acid substitutions that decouple the dependence of the association state on the cluster are shown to have no effect on the overall cluster conversion mechanism but have a significant effect on reaction kinetics.

scholarly journals Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

2015 ◽  
Vol 113 (2) ◽  
pp. 326-331 ◽  
Author(s):  
William H. Hudson ◽  
Bradley R. Kossmann ◽  
Ian Mitchelle S. de Vera ◽  
Shih-Wei Chuo ◽  
Emily R. Weikum ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Amino Acid ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Binding Specificity ◽  
Conformational Space ◽  
Amino Acid Substitutions ◽  
Ancestral Gene ◽  
Response Elements ◽  
Binding Interface

Many genomes contain families of paralogs—proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD’s ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors’ response element-binding specificity were far from the proteins’ DNA-binding interface and interacted epistatically to change the DBD’s function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

Biochemical and In Vivo Characterization of Amino Acid Substitutions in the Runx1 (AML1) Runt Domain Found in FPD/AML, AML M0, and Cleidocranial Dysplasia (CCD) Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 464-464
Author(s):  
Christina J. Matheny ◽  
Takeshi Corpora ◽  
Maren E. Speck ◽  
Ting-Lei Gu ◽  
John H. Bushweller ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Domain Structure ◽  
Cleidocranial Dysplasia ◽  
Amino Acid Substitutions ◽  
Runt Domain ◽  
Core Binding Factor ◽  
Binding Factor

Abstract Runx1 and CBF β are the DNA-binding and non DNA-binding subunits of a core-binding factor that is required for hematopoiesis, and that is frequently mutated in leukemia. Runx2 is the DNA-binding subunit of a core-binding factor required for bone formation. Mono-allelic deletion, nonsense, frameshift, and missense mutations have been found in RUNX1 in familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML) and in myelodysplastic syndrome (MDS), and biallelic mutations in RUNX1 are found in 20% of AML M0 patients. Similar types of mono-allelic mutations have been found in RUNX2 in patients with cleidocranial dysplasia (CCD), an inherited skeletal syndrome. FPD/AML and CCD pedigrees have revealed varying degrees of disease severity depending on the nature of the specific mutation. Additionally, it has been observed that mutations involving amino acids in the DNA binding Runt domain that directly contact DNA are associated primarily with Runx1 and hematopoietic disorders, while mutations predicted to disrupt CBF β binding or the Runt domain structure are found only in Runx2 in CCD patients. We introduced 21 amino acid substitutions into the Runt domain of Runx1 identified in FPD/AML, AML M0, and CCD patients, and quantified their effects on DNA binding, heterodimerization with CBFβ, and the Runt domain structure using yeast one- and two-hybrid, quantitative electrophoretic mobility shift, heteronuclear single quantum correlation spectroscopy, and urea denaturation experiments. To address the impact on in vivo function, several of these point mutations were engineered into the endogenous Runx1 allele in mice. These five mutations include: R177X, R174Q, T149A, T161A, and L148F. R177X is found in FPD/AML patients and truncates Runx1 two amino acids before the C-terminal boundary of the Runt domain. R174Q (found in FPD/AML and CCD) disrupts DNA binding 1000-fold, but does not disrupt CBFb binding or perturb the Runt domain fold. T149A (found only in CCD) disrupts CBFβ binding 13-fold while T161A (not found in patients) disrupts CBFβ binding 40-fold. Both T149A and T161A slightly perturb the Runt domain fold, but do not alter DNA binding affinity. L148F (found in CCD) also disrupts the Runt domain fold, and decreases DNA binding. All animals heterozygous for these alleles are viable. Mice homozygous for R177X and R174Q die during gestation. Mice homozygous for the T149A and T161A mutations, on the other hand, are born at normal Mendelian frequencies, but 62% and 100%, respectively, die by or at three weeks of age from an undetermined cause. The effects of these mutations on hematopoietic progenitor and platelet numbers, both of which are affected in FPD/AML patients, will be presented. We conclude that mutations that affect CBFβ binding result in hypomorphic Runx1 alleles, while mutations involving DNA contacts result in more severe inactivation of Runx1 function. Thus FPD/AML, AML M0, and MDS require mutations that severely inactivate Runx1 function, while CCD can result from more subtle alterations in Runx2.

scholarly journals Creating new DNA binding specificities in the yeast transcriptional activator GCN4 by combining selected amino acid substitutions

1994 ◽  
Vol 22 (12) ◽  
pp. 2198-2208 ◽  
Author(s):  
Manfred Suckow ◽  
Anup Madan ◽  
Brigitte Kisters-Woike ◽  
Brigitte von Wilcken-Bergmann ◽  
Benno Müller-Hill
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activator ◽  
Amino Acid Substitutions

scholarly journals Phenotypic Analysis of Random hnsMutations Differentiate DNA-Binding Activity from Properties offimA Promoter Inversion Modulation and Bacterial Motility

1999 ◽  
Vol 181 (3) ◽  
pp. 941-948 ◽  
Author(s):  
Gina M. Donato ◽  
Thomas H. Kawula
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Constitutive Expression ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Phenotypic Analysis ◽  
Mutator Strain ◽  
Dna Binding Activity ◽  
And Function

ABSTRACT H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of theproU and fimB genes, increased fimApromoter inversion rates, and repression of the flhCDmaster operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proUexpression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimBoccurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hnsmutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.

scholarly journals Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects

Blood ◽  
2013 ◽  
Vol 122 (25) ◽  
pp. 4090-4093 ◽  
Author(s):  
Jacqueline Stockley ◽  
Neil V. Morgan ◽  
Danai Bem ◽  
Gillian C. Lowe ◽  
Marie Lordkipanidzé ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Dense Granule ◽  
Amino Acid Substitutions ◽  
Dna Binding Domain ◽  
Excessive Bleeding ◽  
Key Points ◽  
Dense Granule Secretion ◽  
Granule Secretion

Key Points Novel FLI1 and RUNX1 alterations were identified in 6 of 13 patients with excessive bleeding and platelet granule secretion defects. Two FLI1 alterations predicting amino acid substitutions in the DNA-binding domain of FLI1 abolished transcriptional activity of FLI1.

scholarly journals Mutation Analysis of PobR and PcaU, Closely Related Transcriptional Activators in Acinetobacter

1998 ◽  
Vol 180 (19) ◽  
pp. 5058-5069 ◽  
Author(s):  
Ruben G. Kok ◽  
David A. D’Argenio ◽  
L. Nicholas Ornston
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Primary Structure ◽  
Evolutionary Divergence ◽  
Amino Acid Substitutions ◽  
Transcriptional Activators ◽  
Loss Of Function ◽  
Primary Sequence ◽  
Nucleotide Substitutions

ABSTRACT Acinetobacter PobR and PcaU are transcriptional activators that closely resemble each other in primary structure, DNA-binding sites, metabolic modulators, and physiological function. PobR responds to the inducer-metabolite p-hydroxybenzoate and activates transcription of pobA, the structural gene for the enzyme that converts p-hydroxybenzoate to protocatechuate. This compound, differing fromp-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for protocatechuate catabolism. Particular experimental attention has been paid to PobR and PcaU fromAcinetobacter strain ADP1, which exhibits exceptional competence for natural transformation. This trait allowed selection of mutant strains in which pobR function had been impaired by nucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum of amino acids conserved by the demand for continued function during evolutionary divergence of PobR, PcaU, and related proteins. Surface plasmon resonance was used to determine the ability of mutant PobR proteins to bind to DNA in the pobA-pobR intergenic region. Deleterious mutations that strongly affect DNA binding all cluster in and around the PobR region that contains a helix-turn-helix motif, whereas mutations causing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generated mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on p-hydroxybenzoate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequence. Independent mutations allowing PcaU to mimic PobR activity were shown to be G222V amino acid substitutions in the C terminus of the 274-residue protein. Together, the analyses suggest that PobR and PcaU possess a linear domain structure similar to that of LysR transcriptional activators which largely differ in primary structure.

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