scholarly journals Functional Domains of the Bacillus subtilis Transcription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding

2006 ◽  
Vol 188 (8) ◽  
pp. 3024-3036 ◽  
Author(s):  
Irina Saraiva Franco ◽  
Luís Jaime Mota ◽  
Cláudio Manuel Soares ◽  
Isabel de Sá-Nogueira
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Dna Binding ◽  
Random Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Functional Domains ◽  
Nucleoprotein Complex ◽  
Effector Binding

ABSTRACT The Bacillus subtilis AraR transcription factor represses at least 13 genes required for the extracellular degradation of arabinose-containing polysaccharides, transport of arabinose, arabinose oligomers, xylose, and galactose, intracellular degradation of arabinose oligomers, and further catabolism of this sugar. AraR exhibits a chimeric organization comprising a small N-terminal DNA-binding domain that contains a winged helix-turn-helix motif similar to that seen with the GntR family and a larger C-terminal domain homologous to that of the LacI/GalR family. Here, a model for AraR was derived based on the known crystal structures of the FadR and PurR regulators from Escherichia coli. We have used random mutagenesis, deletion, and construction of chimeric LexA-AraR fusion proteins to map the functional domains of AraR required for DNA binding, dimerization, and effector binding. Moreover, predictions for the functional role of specific residues were tested by site-directed mutagenesis. In vivo analysis identified particular amino acids required for dimer assembly, formation of the nucleoprotein complex, and composition of the sugar-binding cleft. This work presents a structural framework for the function of AraR and provides insight into the mechanistic mode of action of this modular repressor.

scholarly journals A Region of Bacillus subtilis CodY Protein Required for Interaction with DNA

2005 ◽  
Vol 187 (12) ◽  
pp. 4127-4139 ◽  
Author(s):  
Pascale Joseph ◽  
Manoja Ratnayake-Lecamwasam ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Dna Recognition ◽  
Transcriptional Regulators ◽  
Site Directed Mutagenesis ◽  
Regulation Of Transcription ◽  
Gram Positive Bacteria ◽  
Target Promoters

ABSTRACT Bacillus subtilis CodY protein is the best-studied member of a novel family of global transcriptional regulators found ubiquitously in low-G+C gram-positive bacteria. As for many DNA-binding proteins, CodY appears to have a helix-turn-helix (HTH) motif thought to be critical for interaction with DNA. This putative HTH motif was found to be highly conserved in the CodY homologs. Site-directed mutagenesis was used to identify amino acids within this motif that are important for DNA recognition and binding. The effects of each mutation on DNA binding in vitro and on the regulation of transcription in vivo from two target promoters were tested. Each of the mutations had similar effects on binding to the two promoters in vitro, but some mutations had differential effects in vivo.

scholarly journals Sporulation Phenotype of a Bacillus subtilis Mutant Expressing an Unprocessable but Active σE Transcription Factor

2004 ◽  
Vol 186 (7) ◽  
pp. 1999-2005 ◽  
Author(s):  
Shonna McBride ◽  
W. G. Haldenwang
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Stage Iii ◽  
Wild Type ◽  
Developmentally Regulated ◽  
Proprotein Processing ◽  
Residual Inhibition

ABSTRACT σE, a sporulation-specific sigma factor of Bacillus subtilis, is formed from an inactive precursor (pro-σE) by a developmentally regulated processing reaction that removes 27 amino acids from the proprotein's amino terminus. A sigE variant (sigE335) lacking 15 amino acids of the prosequence is not processed into mature σE but is active without processing. In the present work, we investigated the sporulation defect in sigE335-expressing B. subtilis, asking whether it is the bypass of proprotein processing or a residual inhibition of σE activity that is responsible. Fluorescence microscopy demonstrated that sigE335-expressing B. subtilis progresses further into sporulation (stage III) than do strains lacking σE activity (stage II). Consistent with its stage III phenotype, and a defect in σE activity rather than its timing, the sigE335 allele did not disturb early sporulation gene expression but did inhibit the expression of late sporulation genes (gerE and sspE). The Spo− phenotype of sigE335 was found to be recessive to wild-type sigE. In vivo assays of σE activity in sigE, sigE335, and merodiploid strains indicate that the residual prosequence on σE335, still impairs its activity to function as a transcription factor. The data suggest that the 11-amino-acid extension on σE335 allows it to bind RNA polymerase and direct the resulting holoenzyme to σE-dependent promoters but reduces the enzyme's ability to initiate transcription initiation and/or exit from the promoter.

scholarly journals The SKHR Motif Is Required for Biological Function of the VirR Response Regulator from Clostridium perfringens

2003 ◽  
Vol 185 (20) ◽  
pp. 6205-6208 ◽  
Author(s):  
Sheena McGowan ◽  
Jennifer R. O'Connor ◽  
Jackie K. Cheung ◽  
Julian I. Rood
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Clostridium Perfringens ◽  
Biological Function ◽  
Response Regulator ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Toxin Gene

ABSTRACT The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen Clostridium perfringens. The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins. VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain. Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein. Random mutagenesis identified a unique residue, Met-172, that was required for biological function. Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity. Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein.

scholarly journals Mutational Analysis of the MutH Protein fromEscherichia coli

2000 ◽  
Vol 276 (15) ◽  
pp. 12113-12119 ◽  
Author(s):  
Tamalette Loh ◽  
Kenan C. Murphy ◽  
Martin G. Marinus
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Flexible Loop ◽  
Loop Regions ◽  
Binding Residues

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH andPvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis asPvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops inPvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) inPvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity bothin vivoandin vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHΔ224 and MutHΔ214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).

scholarly journals Genetic and Biochemical Analysis of the Interaction of Bacillus subtilis CodY with Branched-Chain Amino Acids

2009 ◽  
Vol 191 (22) ◽  
pp. 6865-6876 ◽  
Author(s):  
Anuradha C. Villapakkam ◽  
Luke D. Handke ◽  
Boris R. Belitsky ◽  
Vladimir M. Levdikov ◽  
Anthony J. Wilkinson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Branched Chain Amino Acids ◽  
Site Directed Mutagenesis ◽  
Binding Studies ◽  
Hydrophobic Pocket ◽  
Isoleucine Leucine ◽  
Branched Chain

ABSTRACT Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

scholarly journals Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

2021 ◽  
Vol 49 (7) ◽  
pp. 3856-3875
Author(s):  
Marina Kulik ◽  
Melissa Bothe ◽  
Gözde Kibar ◽  
Alisa Fuchs ◽  
Stefanie Schöne ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Dna Binding ◽  
Receptor Binding ◽  
Binding Sites ◽  
Specific Gene ◽  
Functional Diversification ◽  
Enhancer Activity ◽  
Sequence Composition

Abstract The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor

1986 ◽  
Vol 6 (12) ◽  
pp. 4723-4733
Author(s):  
L A Chodosh ◽  
R W Carthew ◽  
P A Sharp
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Affinity Column ◽  
Dna Binding Activity ◽  
Dna Fragment

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

scholarly journals Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

1998 ◽  
Vol 18 (10) ◽  
pp. 5670-5677 ◽  
Author(s):  
Ossama Abu Hatoum ◽  
Shlomit Gross-Mesilaty ◽  
Kristin Breitschopf ◽  
Aviad Hoffman ◽  
Hedva Gonen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
26S Proteasome ◽  
Intact Cells ◽  
Free System ◽  
Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

scholarly journals Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif

1993 ◽  
Vol 13 (1) ◽  
pp. 123-132
Author(s):  
A D Sharrocks ◽  
H Gille ◽  
P E Shaw
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

scholarly journals Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

2008 ◽  
Vol 190 (18) ◽  
pp. 6134-6147 ◽  
Author(s):  
Shigeo Tojo ◽  
Takenori Satomura ◽  
Kanako Kumamoto ◽  
Kazutake Hirooka ◽  
Yasutaro Fujita
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Stringent Response ◽  
Branched Chain Amino Acids ◽  
Base Substitution ◽  
Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

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