scholarly journals The Structure of Bradyrhizobium japonicum Transcription Factor FixK2 Unveils Sites of DNA Binding and Oxidation

2013 ◽  
Vol 288 (20) ◽  
pp. 14238-14246 ◽  
Author(s):  
Mariette Bonnet ◽  
Mareike Kurz ◽  
Socorro Mesa ◽  
Christophe Briand ◽  
Hauke Hennecke ◽  
...  
Keyword(s):  
Dna Binding ◽  
Bradyrhizobium Japonicum ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Receptor Protein ◽  
Target Sequence ◽  
Protein Variant ◽  
Life Styles ◽  
C Terminus ◽  
Camp Receptor

FixK2 is a regulatory protein that activates a large number of genes for the anoxic and microoxic, endosymbiotic, and nitrogen-fixing life styles of the α-proteobacterium Bradyrhizobium japonicum. FixK2 belongs to the cAMP receptor protein (CRP) superfamily. Although most CRP family members are coregulated by effector molecules, the activity of FixK2 is negatively controlled by oxidation of its single cysteine (Cys-183) located next to the DNA-binding domain and possibly also by proteolysis. Here, we report the three-dimensional x-ray structure of FixK2, a representative of the FixK subgroup of the CRP superfamily. Crystallization succeeded only when (i) an oxidation- and protease-insensitive protein variant (FixK2(C183S)-His6) was used in which Cys-183 was replaced with serine and the C terminus was fused with a hexahistidine tag and (ii) this protein was allowed to form a complex with a 30-mer double-stranded target DNA. The structure of the FixK2-DNA complex was solved at a resolution of 1.77 Å, at which the protein formed a homodimer. The precise protein-DNA contacts were identified, which led to an affirmation of the canonical target sequence, the so-called FixK2 box. The C terminus is surface-exposed, which might explain its sensitivity to specific cleavage and degradation. The oxidation-sensitive Cys-183 is also surface-exposed and in close proximity to DNA. Therefore, we propose a mechanism whereby the oxo acids generated after oxidation of the cysteine thiol cause an electrostatic repulsion, thus preventing specific DNA binding.

A multitude of CRP/FNR-like transcription proteins in Bradyrhizobium japonicum

2006 ◽  
Vol 34 (1) ◽  
pp. 156-159 ◽  
Author(s):  
S. Mesa ◽  
H. Hennecke ◽  
H.-M. Fischer
Keyword(s):  
Gene Expression ◽  
Bradyrhizobium Japonicum ◽  
Sequence Similarity ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Low Oxygen ◽  
Denitrification Gene ◽  
Camp Receptor

In Bradyrhizobium japonicum, the nitrogen-fixing soya bean endosymbiont and facultative denitrifier, three CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein)-type transcription factors [FixK1, FixK2 and NnrR (nitrite and nitric oxide reductase regulator)] have been studied previously in the context of the regulation of nitrogen fixation and denitrification. The gene expression of both fixK1 and nnrR depends on FixK2, which acts as a key distributor of the ‘low-oxygen’ signal perceived by the two-component regulatory system FixLJ. While the targets for FixK1 are not known, NnrR transduces the nitrogen oxide signal to the level of denitrification gene expression. Besides these three regulators, the complete genome sequence of this organism has revealed the existence of 13 additional CRP/FNR-type proteins whose functions have not yet been studied. Based on sequence similarity and phylogenetic analysis, we discuss in this paper the peculiarities of these additional factors.

Site-directed mutants of the cAMP receptor protein-DNA binding of five mutant proteins

1987 ◽  
Vol 1 (3) ◽  
pp. 201-203 ◽  
Author(s):  
Manda E. Gent ◽  
Silvia Gärtner ◽  
Angela M. Gronenborn ◽  
Rodica Sandulache ◽  
G.Marius Clore
Keyword(s):  
Dna Binding ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Mutant Proteins ◽  
Camp Receptor

scholarly journals The DNA Architectural Protein HMGB1 Displays Two Distinct Modes of Action That Promote Enhanceosome Assembly

2002 ◽  
Vol 22 (12) ◽  
pp. 4390-4401 ◽  
Author(s):  
Katherine Mitsouras ◽  
Ben Wong ◽  
Charina Arayata ◽  
Reid C. Johnson ◽  
Michael Carey
Keyword(s):  
Dna Binding ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Specific Interaction ◽  
Regulatory Region ◽  
Direct Interaction ◽  
Single Amino Acid ◽  
Target Sequence ◽  
Mobility Shift ◽  
Epstein Barr

ABSTRACT HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.

scholarly journals LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2650-2660 ◽  
Author(s):  
Hideaki Takano ◽  
Yoshihiro Agari ◽  
Kenta Hagiwara ◽  
Ren Watanabe ◽  
Ryuta Yamazaki ◽  
...  
Keyword(s):  
Thermus Thermophilus ◽  
Regulatory Protein ◽  
Carotenoid Biosynthesis ◽  
Thermophilic Bacterium ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
S1 Nuclease ◽  
Positive Regulator ◽  
Camp Receptor

LdrP (TT_P0055) (LitR-dependent regulatory protein) is one of the four cAMP receptor protein (CRP)/FNR family transcriptional regulators retained by the extremely thermophilic bacterium Thermus thermophilus. Previously, we reported that LdrP served as a positive regulator for the light-induced transcription of crtB, a carotenoid biosynthesis gene encoded on the megaplasmid of this organism. Here, we showed that LdrP also functions as an activator of the expression of genes clustered around the crtB gene under the control of LitR, an adenosyl B12-bound light-sensitive regulator. Transcriptome analysis revealed the existence of 19 LitR-dependent genes on the megaplasmid. S1 nuclease protection assay confirmed that the promoters preceding TT_P0044 (P44), TT_P0049 (P49) and TT_P0070 (P70) were activated upon illumination in the WT strain. An ldrP mutant lost the ability to activate P44, P49 and P70, whilst disruption of litR resulted in constitutive transcription from these promoters irrespective of illumination, indicating that these genes were photo-dependently regulated by LdrP and LitR. An in vitro transcription experiment demonstrated that LdrP directly activated mRNA synthesis from P44 and P70 by the Thermus RNA polymerase holocomplex. The present evidence indicated that LdrP was the positive regulator essential for the transcription of the T. thermophilus light-inducible cluster encoded on the megaplasmid.

scholarly journals Systematic single base-pair substitution analysis of DNA binding by the cAMP receptor protein in cyanobacteriumSynechocystissp. PCC 6803

FEBS Letters ◽  
2004 ◽  
Vol 563 (1-3) ◽  
pp. 55-58 ◽  
Author(s):  
Katsumi Omagari ◽  
Hidehisa Yoshimura ◽  
Mitsunori Takano ◽  
Dongyun Hao ◽  
Masayuki Ohmori ◽  
...  
Keyword(s):  
Dna Binding ◽  
Base Pair ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Single Base ◽  
Single Base Pair Substitution ◽  
Single Base Pair ◽  
Base Pair Substitution ◽  
Camp Receptor ◽  
Pcc 6803

Point mutations in the DNA- and cNMP-binding domains of the homologue of the cAMP receptor protein (CRP) in Mycobacterium bovis BCG: implications for the inactivation of a global regulator and strain attenuation

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 547-556 ◽  
Author(s):  
Claire L. Spreadbury ◽  
Mark J. Pallen ◽  
Tim Overton ◽  
Marcel A. Behr ◽  
Serge Mostowy ◽  
...  
Keyword(s):  
Dna Binding ◽  
Mycobacterium Bovis ◽  
Point Mutations ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Global Regulator ◽  
Reporter System ◽  
Binding Domains ◽  
Camp Receptor

The genome of Mycobacterium tuberculosis H37Rv includes a homologue of the CRP/FNR (cAMP receptor protein/fumarate and nitrate reduction regulator) family of transcription regulators encoded by Rv3676. Sequencing of the orthologous gene from attenuated Mycobacterium bovis Bacille Calmette–Guérin (BCG) strains revealed point mutations that affect the putative DNA-binding and cNMP-binding domains of the encoded protein. These mutations are not present in the published sequences of the Rv3676 orthologues in M. bovis, M. tuberculosis or Mycobacterium leprae. An Escherichia coli lacZ reporter system was used to show that the M. tuberculosis Rv3676 protein binds to DNA sites for CRP, but this DNA binding was decreased or abolished with the Rv3676 protein counterparts from BCG strains. The DNA-binding ability of the M. tuberculosis Rv3676 protein was decreased by the introduction of base changes corresponding to the BCG point mutations. Conversely, the DNA binding of the BCG Rv3676 proteins from BCG strains was restored by removing the mutations. These data show that in this reporter system the point mutations present in the Rv3676 orthologue in BCG strains render its function defective (early strains) or abolished (late strains) and suggest that this protein might be naturally defective in M. bovis BCG strains. This raises the possibility that a contributing factor to the attenuation of BCG strains may be an inability of this global regulator to control the expression of genes required for in vivo survival and persistence.

scholarly journals DNA Binding modes of cAMP receptor protein(CRP) : difference between CRP-(cAMP)_1 and CRP-(cAMP)_2

2001 ◽  
Vol 41 (supplement) ◽  
pp. S183
Author(s):  
A. Matsubuchi ◽  
N. Fujimoto ◽  
A. Toyama ◽  
H. Takeuchi
Keyword(s):  
Dna Binding ◽  
Receptor Protein ◽  
Binding Modes ◽  
Camp Receptor Protein ◽  
Camp Receptor

Comparison of cAMP receptor protein (CRP) and a cAMP-independent form of CRP by Raman spectroscopy and DNA binding

Biochemistry ◽  
1991 ◽  
Vol 30 (20) ◽  
pp. 5076-5080 ◽  
Author(s):  
Guo Shen Tan ◽  
Patrick Kelly ◽  
Jin Kim ◽  
Roger M. Wartell
Keyword(s):  
Raman Spectroscopy ◽  
Dna Binding ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Independent Form ◽  
Camp Receptor
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