Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor

The competence transcription factor ComK is the master regulator of competence development in Bacillus subtilis. In the regulatory pathway, ComK is involved in different interactions: (i) protein–DNA interactions to stimulate transcription of ComK-dependent genes and (ii) protein–protein interactions, divided into interactions with other proteins and interactions between ComK proteins involving oligomerization. The fact that ComK displays different types of interactions suggests the presence of specific, distinct domains in the protein. This paper describes a search for functional domains, by constructing ComK truncation variants, which were tested for DNA binding, oligomerization and transcription activation. Truncations at the C-terminal end of ComK demonstrated the requirement of this part for transcription activation, but not for DNA binding. The C-terminal region is probably involved in oligomerization of ComK-dimers into tetramers. Surprisingly, a ComK truncation variant lacking 9 aa from the N-terminal end (ΔN9ComK) showed higher transcription activation than wild-type ComK, when expressed in Lactococcus lactis. However, in B. subtilis, transcription activation by ΔN9ComK was twofold lower than that by wild-type ComK, resulting from a five- to sixfold lower protein level of ComKΔN9. Thus, relatively, ΔN9ComK is more active in transcription activation than wild-type ComK. These results suggest that the presence of this N-terminal extension on ComK is a trade-off between high transcription activation and a thus far unidentified role in regulation of ComK.

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

Journal of Bacteriology ◽

10.1128/jb.188.8.3024-3036.2006 ◽

2006 ◽

Vol 188 (8) ◽

pp. 3024-3036 ◽

Cited By ~ 22

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.

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Two Regions of Bacillus subtilis Transcription Factor SpoIIID Allow a Monomer To Bind DNA

Journal of Bacteriology ◽

10.1128/jb.01506-09 ◽

2010 ◽

Vol 192 (6) ◽

pp. 1596-1606 ◽

Cited By ~ 5

Author(s):

Paul Himes ◽

Steven J. McBryant ◽

Lee Kroos

Keyword(s):

Transcription Factor ◽

Bacillus Subtilis ◽

Dna Binding ◽

Transcriptional Activation ◽

Binding Sites ◽

Mother Cell ◽

Consensus Sequence ◽

Human Pathogens ◽

High Affinity ◽

Basic Region

ABSTRACT Nutrient limitation causes Bacillus subtilis to develop into two different cell types, a mother cell and a spore. SpoIIID is a key regulator of transcription in the mother cell and positively or negatively regulates more than 100 genes, in many cases by binding to the promoter region. SpoIIID was predicted to have a helix-turn-helix motif for sequence-specific DNA binding, and a 10-bp consensus sequence was recognized in binding sites, but some strong binding sites were observed to contain more than one match to the consensus sequence, suggesting that SpoIIID might bind as a dimer or cooperatively as monomers. Here we show that SpoIIID binds with high affinity as a monomer to a single copy of its recognition sequence. Using charge reversal substitutions of residues likely to be exposed on the surface of SpoIIID and assays for transcriptional activation in vivo and for DNA binding in vitro, we identify two regions essential for DNA binding, the putative recognition helix of the predicted helix-turn-helix motif and a basic region near the C terminus. SpoIIID is unusual among prokaryotic DNA-binding proteins with a single helix-turn-helix motif in its ability to bind DNA monomerically with high affinity. We propose that the C-terminal basic region of SpoIIID makes additional contacts with DNA, analogous to the N-terminal arm of eukaryotic homeodomain proteins and the “wings” of winged-helix proteins, but structurally distinct. SpoIIID is highly conserved only among bacteria that form endospores, including several important human pathogens. The need to conserve biosynthetic capacity during endospore formation might have favored the evolution of a small transcription factor capable of high-affinity binding to DNA as a monomer, and this unusual mode of DNA binding could provide a target for drug design.

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C-terminus of transcription factor TnrA from Bacillus subtilis controls DNA-binding domain activity but is not required for dimerization

Molecular Biology ◽

10.1134/s0026893313020052 ◽

2013 ◽

Vol 47 (2) ◽

pp. 293-298 ◽

Cited By ~ 2

Author(s):

K. P. Fedorova ◽

I. S. Scharafutdinov ◽

E. Y. Turbina ◽

M. I. Bogachev ◽

O. N. Ilinskaja ◽

...

Keyword(s):

Transcription Factor ◽

Bacillus Subtilis ◽

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain ◽

C Terminus ◽

Transcription Factor Tnra

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A Single, Specific Thymine Mutation in the ComK-Binding Site Severely Decreases Binding and Transcription Activation by the Competence Transcription Factor ComK of Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00281-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4718-4728 ◽

Cited By ~ 8

Author(s):

Kim A. Susanna ◽

Aleksandra M. Mironczuk ◽

Wiep Klaas Smits ◽

Leendert W. Hamoen ◽

Oscar P. Kuipers

Keyword(s):

Transcription Factor ◽

Bacillus Subtilis ◽

Dna Binding ◽

Consensus Sequence ◽

Transcription Activation ◽

Specific Sequence ◽

Flexible Spacer ◽

Specific Position ◽

Dna Binding Affinity ◽

In Silico Analyses

ABSTRACT The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating the transcription of the K regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region. Each K-box consists of two AT-boxes with the consensus sequence AAAA-(N)5-TTTT, which are separated by a flexible spacer resulting in either two, three, or four helical turns between the starting nucleotides of the repeating AT-box units. In this study, the effects of potential determinants of ComK regulation in K-boxes were investigated by testing ComK's transcription activation and DNA-binding affinity on altered K-boxes with mutations either in the spacer between the AT-boxes or in the consensus sequence of the AT-boxes. The most striking result demonstrates the importance of the second thymine base in the AT-boxes. Mutation of this T into a guanine resulted in a threefold reduction in transcription activation and DNA binding by ComK. Transcription activation, as well as DNA binding, was almost completely abolished when both AT-boxes contained a T2-to-G mutation. This result was corroborated by in silico analyses demonstrating that a combination of mutations at the T2 positions of both AT-boxes is not found among any ComK-activated K-boxes, indicating that at least one consensus T2 position is required to maintain a functional K-box. The results suggest an important structural role for T2 in ComK binding, probably by its specific position in the minor groove of the DNA.

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