Alanine substitution mutations in the DNA binding region of a global staphylococcal virulence regulator affect its structure, function, and stability

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.

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Structure/Function Relationships in the Bacteriophage T4 Single-Stranded DNA-Binding Protein

Molecular Biology of Chromosome Function ◽

10.1007/978-1-4612-3652-8_14 ◽

1989 ◽

pp. 302-322

Author(s):

Yousif Shamoo ◽

Kathleen M. Keating ◽

Kenneth R. Williams ◽

William H. Konigsberg

Keyword(s):

Dna Binding ◽

Structure Function ◽

Binding Protein ◽

Bacteriophage T4 ◽

Dna Binding Protein ◽

Single Stranded Dna

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Structure, function, and dynamics of the dimerization and DNA-binding domain of oncogenic transcription factor v-Myc11Edited by P. E. Wright

Journal of Molecular Biology ◽

10.1006/jmbi.2001.4537 ◽

2001 ◽

Vol 307 (5) ◽

pp. 1395-1410 ◽

Cited By ~ 70

Author(s):

Wolfgang Fieber ◽

Martin L. Schneider ◽

Theresia Matt ◽

Bernhard Kräutler ◽

Robert Konrat ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Structure Function ◽

Binding Domain ◽

Factor V ◽

Dna Binding Domain ◽

Oncogenic Transcription Factor

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Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.6.4.1218-1227.1986 ◽

1986 ◽

Vol 6 (4) ◽

pp. 1218-1227

Author(s):

L Naumovski ◽

E C Friedberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Excision Repair ◽

Random Mutagenesis ◽

Nucleotide Binding ◽

Yeast Cells ◽

Wild Type ◽

Binding Region ◽

Site Specific ◽

Essential Function

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.

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Free Fatty Acids Interfere with the DNA Binding Activity of the Virulence Regulator PrfA of Listeria monocytogenes

Journal of Bacteriology ◽

10.1128/jb.00156-20 ◽

2020 ◽

Vol 202 (15) ◽

Cited By ~ 1

Author(s):

Patrícia T. dos Santos ◽

Rikke S. S. Thomasen ◽

Mathias S. Green ◽

Nils J. Færgeman ◽

Birgitte H. Kallipolitis

Keyword(s):

Fatty Acids ◽

Dna Binding ◽

Free Fatty Acids ◽

Antimicrobial Agents ◽

Virulence Gene ◽

Binding Activity ◽

Content Type ◽

Dna Binding Activity ◽

Virulence Regulator ◽

Long Chain

ABSTRACT Naturally occurring free fatty acids (FFAs) are recognized as potent antimicrobial agents that also affect the production of virulence factors in bacterial pathogens. In the foodborne pathogen Listeria monocytogenes, some medium- and long-chain FFAs act as antimicrobial agents as well as signaling compounds, causing a repression of transcription of virulence genes. We previously observed that the master virulence regulator PrfA is involved in both the antimicrobial and virulence-inhibitory response of L. monocytogenes to selected FFAs, but the underlying mechanisms are presently unknown. Here, we present a systematic analysis of the antimicrobial and PrfA-inhibitory activities of medium- and long-chain FFAs of various carbon chain lengths and degrees of saturation. We observed that exposure to specific antimicrobial and nonantimicrobial FFAs prevented PrfA-dependent activation of virulence gene transcription and reduced the levels of PrfA-regulated virulence factors. Thus, an antimicrobial activity was not compulsory for the PrfA-inhibitory ability of an FFA. In vitro binding experiments revealed that PrfA-inhibitory FFAs were also able to prevent the constitutively active variant PrfA* from binding to the PrfA box in the promoter region of the virulence gene hly, whereas noninhibitory FFAs did not affect its ability to bind DNA. Notably, the unsaturated FFAs inhibited the DNA binding activity of PrfA* most efficiently. Altogether, our findings support a model in which specific FFAs orchestrate a generalized reduction of the virulence potential of L. monocytogenes by directly targeting the key virulence regulator PrfA. IMPORTANCE Listeria monocytogenes is a Gram-positive pathogen able to cause foodborne infections in humans and animals. Key virulence genes in L. monocytogenes are activated by the transcription regulator PrfA, a DNA binding protein belonging to the CRP/FNR family. Various signals from the environment are known to affect the activity of PrfA, either positively or negatively. Recently, we found that specific medium- and long-chain free fatty acids act as antimicrobial agents as well as signaling compounds in L. monocytogenes. Here, we show that both antimicrobial and nonantimicrobial free fatty acids inhibit PrfA-dependent activation of virulence gene transcription by interfering with the DNA binding activity of PrfA. Our findings suggest that free fatty acids could be candidates for alternative therapies against L. monocytogenes.

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Rgg protein structure–function and inhibition by cyclic peptide compounds

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1500357112 ◽

2015 ◽

Vol 112 (16) ◽

pp. 5177-5182 ◽

Cited By ~ 46

Author(s):

Vijay Parashar ◽

Chaitanya Aggarwal ◽

Michael J. Federle ◽

Matthew B. Neiditch

Keyword(s):

Dna Binding ◽

Structure Function ◽

Cyclosporin A ◽

Crystal Structures ◽

Disulfide Bond ◽

Binding Domain ◽

Dna Binding Domain ◽

X Ray ◽

Peptide Pheromone ◽

Dimerization Interface

Peptide pheromone cell–cell signaling (quorum sensing) regulates the expression of diverse developmental phenotypes (including virulence) in Firmicutes, which includes common human pathogens, e.g.,Streptococcus pyogenesandStreptococcus pneumoniae. Cytoplasmic transcription factors known as “Rgg proteins” are peptide pheromone receptors ubiquitous in Firmicutes. Here we present X-ray crystal structures of aStreptococcusRgg protein alone and in complex with a tight-binding signaling antagonist, the cyclic undecapeptide cyclosporin A. To our knowledge, these represent the first Rgg protein X-ray crystal structures. Based on the results of extensive structure–function analysis, we reveal the peptide pheromone-binding site and the mechanism by which cyclosporin A inhibits activation of the peptide pheromone receptor. Guided by the Rgg–cyclosporin A complex structure, we predicted that the nonimmunosuppressive cyclosporin A analog valspodar would inhibit Rgg activation. Indeed, we found that, like cyclosporin A, valspodar inhibits peptide pheromone activation of conserved Rgg proteins in medically relevantStreptococcusspecies. Finally, the crystal structures presented here revealed that the Rgg protein DNA-binding domains are covalently linked across their dimerization interface by a disulfide bond formed by a highly conserved cysteine. The DNA-binding domain dimerization interface observed in our structures is essentially identical to the interfaces previously described for other members of the XRE DNA-binding domain family, but the presence of an intermolecular disulfide bond buried in this interface appears to be unique. We hypothesize that this disulfide bond may, under the right conditions, affect Rgg monomer–dimer equilibrium, stabilize Rgg conformation, or serve as a redox-sensitive switch.

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