scholarly journals Gene Replacement Analysis of the Streptomyces virginiae barA Gene Encoding the Butyrolactone Autoregulator Receptor Reveals that BarA Acts as a Repressor in Virginiamycin Biosynthesis

1998 ◽  
Vol 180 (13) ◽  
pp. 3317-3322 ◽  
Author(s):  
Hiroko Nakano ◽  
Emio Takehara ◽  
Takuya Nihira ◽  
Yasuhiro Yamada
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
Regulatory Protein ◽  
Gene Cassette ◽  
Gene Replacement ◽  
Binding Motif ◽  
Wild Type ◽  
Streptomyces Virginiae ◽  
In The Wild

ABSTRACT Virginiae butanolides (VBs), which are among the butyrolactone autoregulators of Streptomyces species, act as a primary signal in Streptomyces virginiae to trigger virginiamycin biosynthesis and possess a specific binding protein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to virginiamycin biosynthesis, two barAmutant strains (strains NH1 and NH2) were created by homologous recombination. In strain NH1, an internal 99-bp EcoT14I fragment of barA was deleted, resulting in an in-frame deletion of 33 amino acid residues, including the second helix of the probable helix-turn-helix DNA-binding motif. With the same growth rate as wild-type S. virginiae on both solid and liquid media, strain NH1 showed no apparent changes in its morphological behavior, indicating that the VB-BarA pathway does not participate in morphological control in S. virginiae. In contrast, virginiamycin production started 6 h earlier in strain NH1 than in the wild-type strain, demonstrating for the first time that BarA is actively engaged in the control of virginiamycin production and implying that BarA acts as a repressor in virginiamycin biosynthesis. In strain NH2, an internal EcoNI-SmaI fragment of barA was replaced with a divergently oriented neomycin resistance gene cassette, resulting in the C-terminally truncated BarA retaining the intact helix-turn-helix motif. In strain NH2 and in a plasmid-integrated strain containing both intact and mutatedbarA genes, virginiamycin production was abolished irrespective of the presence of VB, suggesting that the mutated BarA retaining the intact DNA-binding motif was dominant over the wild-type BarA. These results further support the hypothesis that BarA works as a repressor in virginiamycin production and suggests that the helix-turn-helix motif is essential to its function. In strain NH1, VB production was also abolished, thus indicating that BarA is a pleiotropic regulatory protein controlling not only virginiamycin production but also autoregulator biosynthesis.

scholarly journals Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans.

1990 ◽  
Vol 10 (6) ◽  
pp. 3194-3203 ◽  
Author(s):  
A Andrianopoulos ◽  
M J Hynes
Keyword(s):  
Dna Binding ◽  
Aspergillus Nidulans ◽  
Nuclear Localization ◽  
Regulatory Gene ◽  
Transcription Activation ◽  
Binding Motif ◽  
Wild Type ◽  
Amino Terminal ◽  
Carboxyl Terminal

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.

scholarly journals Involvement of H-NS in Transpositional Recombination Mediated by IS1

2001 ◽  
Vol 183 (8) ◽  
pp. 2476-2484 ◽  
Author(s):  
Yasuyuki Shiga ◽  
Yasuhiko Sekine ◽  
Yasunobu Kano ◽  
Eiichi Ohtsubo
Keyword(s):  
Dna Binding ◽  
Wild Type Strain ◽  
Integration Host Factor ◽  
Deficient Mutant ◽  
Wild Type ◽  
Transposase Gene ◽  
In The Wild ◽  
Transposition Reaction ◽  
Target Plasmid

ABSTRACT IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter- and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N- and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NS-deficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.

scholarly journals The Nonessential H2A N-Terminal Tail Can Function as an Essential Charge Patch on the H2A.Z Variant N-Terminal Tail

2003 ◽  
Vol 23 (8) ◽  
pp. 2778-2789 ◽  
Author(s):  
Qinghu Ren ◽  
Martin A. Gorovsky
Keyword(s):  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Wild Type Protein ◽  
Lysine Residues ◽  
In The Wild ◽  
Essential Function

ABSTRACT Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up ∼80% of total H2A, and a conserved variant, H2A.Z. We showed previously that acetylation of H2A.Z was essential (Q. Ren and M. A. Gorovsky, Mol. Cell 7:1329-1335, 2001). Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo. Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein. Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential. Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail. Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

scholarly journals Neural network modeling of differential binding between wild-type and mutant CTCF reveals putative binding preferences for zinc fingers 1-2

2021 ◽  
Author(s):  
Irene Miriam Kaplow ◽  
Abhimanyu Banerjee ◽  
Chuan-Sheng Foo
Keyword(s):  
Neural Network ◽  
Dna Binding ◽  
Zinc Fingers ◽  
Neural Network Modeling ◽  
Binding Motif ◽  
Wild Type ◽  
Core Motif ◽  
Binding Motifs ◽  
Binding Domains

Background: Many transcription factors (TFs), such as multi zinc-finger (ZF) TFs, have multiple DNA binding domains (DBDs) with multiple components, and deciphering the DNA binding motifs of individual components is a major challenge. One example of such a TF is CCCTC-binding factor (CTCF), a TF with eleven ZFs that plays a variety of roles in transcriptional regulation, most notably anchoring DNA loops. Previous studies found that CTCF zinc fingers (ZFs) 3-7 bind CTCF's core motif and ZFs 9-11 bind a specific upstream motif, but the motifs of ZFs 1-2 have yet to be identified. Results: We developed a new approach to identifying the binding motifs of individual DBDs of a TF through analyzing chromatin immunoprecipitation sequencing (ChIP-seq) experiments in which a single DBD is mutated: we train a deep convolutional neural network to predict whether wild-type TF binding sites are preserved in the mutant TF dataset and interpret the model. We applied this approach to mouse CTCF ChIP-seq data and, in addition to identifying the known binding preferences of CTCF ZFs 3-11, we identified a GAG binding motif for ZF1 and a weak ATT binding motif for ZF2. We analyzed other CTCF datasets to provide additional evidence that ZFs 1-2 interact with the motifs we identified, and we found that the presence of the motif for ZF1 is associated with Ctcf peak strength. Conclusions: Our approach can be applied to any TF for which in vivo binding data from both the wild-type and mutated versions of the TF are available, and our findings provide an unprecedently comprehensive understanding of the binding preferences of CTCF's DBDs.

scholarly journals DNA Bending by AraC: a Negative Mutant

1998 ◽  
Vol 180 (16) ◽  
pp. 4227-4232 ◽  
Author(s):  
Beatrice Saviola ◽  
Robert R. Seabold ◽  
Robert F. Schleif
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Dna Bending ◽  
Regulatory Protein ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dna Binding Domain ◽  
Wild Type

ABSTRACT We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of Escherichia coli that allows the protein to bind DNA normally but not activate transcription. The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were trans dominant negative with regard to wild-type AraC protein. The mutant with the lowest transcription activation of the araBAD promoter was studied further. It proved to alter a residue that had previously been demonstrated to contact DNA. Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding. Using the phase-sensitive DNA bending assay, we found that wild-type AraC bends DNA about 90° whereas the mutant bends DNA by a smaller amount.

Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans

1990 ◽  
Vol 10 (6) ◽  
pp. 3194-3203
Author(s):  
A Andrianopoulos ◽  
M J Hynes
Keyword(s):  
Dna Binding ◽  
Aspergillus Nidulans ◽  
Nuclear Localization ◽  
Regulatory Gene ◽  
Transcription Activation ◽  
Binding Motif ◽  
Wild Type ◽  
Amino Terminal ◽  
Carboxyl Terminal

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

scholarly journals MyD88-Dependent Signaling Affects the Development of Meningococcal Sepsis by Nonlipooligosaccharide Ligands

2006 ◽  
Vol 74 (6) ◽  
pp. 3538-3546 ◽  
Author(s):  
Laura Plant ◽  
Hong Wan ◽  
Ann-Beth Jonsson
Keyword(s):  
Inflammatory Responses ◽  
Innate Immune ◽  
Meningococcal Sepsis ◽  
Wild Type ◽  
Microbial Infections ◽  
In The Wild ◽  
Myeloid Differentiation Factor ◽  
Dependent Pathway ◽  
Meningococcal Strain

ABSTRACT The Toll-like receptors (TLRs) and the adaptor myeloid differentiation factor 88 (MyD88) are important in the innate immune defenses of the host to microbial infections. Meningococcal ligands signaling via TLRs control inflammatory responses, and stimulation can result in fulminant meningococcal sepsis. In this study, we show that the responses to nonlipooligosaccharide (non-LOS) ligands of meningococci are MyD88 dependent. An isogenic LOS-deficient mutant of the serogroup C meningococcal strain FAM20 caused fatal disease in wild type C57BL/6 mice that was not observed in MyD88−/− mice. Fatality correlated with high proinflammatory cytokine and C5a levels in serum, high neutrophil numbers in blood, and increased bacteremia at 24 h postinfection in the wild-type mice. Infection with the parent strain FAM20 resulted in fatality in 100% of the wild-type mice and 50% of the MyD88−/− mice. We conclude that both LOS and another neisserial ligand cause meningococcal sepsis in an in vivo mouse model and confirm that meningococcal LOS can act via both the MyD88- dependent and -independent pathways, while the non-LOS meningococcal ligand(s) acts only via the MyD88-dependent pathway.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

Effect offurmutation on acid-tolerance response and in vivo virulence of avian septicemicEscherichia coli

2002 ◽  
Vol 48 (5) ◽  
pp. 458-462 ◽  
Author(s):  
Chengru Zhu ◽  
Musangu Ngeleka ◽  
Andrew A Potter ◽  
Brenda J Allan
Keyword(s):  
Parent Strain ◽  
Acid Tolerance ◽  
Wild Type ◽  
Acid Tolerance Response ◽  
E Coli ◽  
Regulator Protein ◽  
In The Wild ◽  
Tolerance Response ◽  
Wild Type Parent

The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria. In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined. The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain. However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation. These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E. coli, as detected by the ability to cause septicemia in our experimental infection.Key words: E. coli, fur, acid-tolerance response.

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