Dataset Information

BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.

ABSTRACT: The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.

SUBMITTER: Bandera AM

PROVIDER: S-EPMC8517857 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The REB1 gene encodes a DNA-binding protein (Reb1p) that is essential for growth of the yeast Saccharomyces cerevisiae. Reb1p binds to sites within transcriptional control regions of genes transcribed by either RNA polymerase I or RNA polymerase II. The sequence of REB1 predicts a protein of 809 amino acids. To define the DNA-binding domain of Reb1p, a series of 5' and 3' deletions within the coding region was constructed in a bacterial expression vector. Analysis of the truncated Reb1p proteins revealed that nearly 400 amino acids of the C-terminal portion of the protein are required for maximal DNA-binding activity. To further define the important structural features of Reb1p, the REB1 homolog from a related yeast, Kluyveromyces lactis, was cloned by genetic complementation. The K. lactis REB1 gene supports active growth of an S. cerevisiae strain whose REB1 gene has been deleted. The Reb1p proteins of the two organisms generate almost identical footprints on DNA, yet the K. lactis REB1 gene encodes a polypeptide of only 595 amino acids. Comparison of the two Reb1p sequences revealed that within the region necessary for the binding of Reb1p to DNA were two long regions of nearly perfect identity, separated in the S. cerevisiae Reb1p by nearly 150 amino acids but in the K. lactis Reb1p by only 40 amino acids. The first includes a 105-amino-acid region related to the DNA-binding domain of the myb oncoprotein; the second bears a faint resemblance to myb. The hypothesis that the DNA-binding domain of Reb1p is formed from these two conserved regions was confirmed by deletion of as many as 90 amino acids between them, with little effect on the DNA-binding ability of the resultant protein. We suggest that the DNA-binding domain of Reb1p is made up of two myb-like regions that, unlike myb itself, are separated by as many as 150 amino acids. Since Reb1p protects only 15 to 20 nucleotides in a chemical or enzymatic footprint assay, the protein must fold such that the two components of the binding site are adjacent.

Dataset Information

BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets