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An improved bind-n-seq strategy to determine protein-DNA interactions validated using the bacterial transcriptional regulator YipR.
- Source :
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BMC microbiology [BMC Microbiol] 2020 Jan 02; Vol. 20 (1), pp. 1. Date of Electronic Publication: 2020 Jan 02. - Publication Year :
- 2020
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Abstract
- Background: Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein-DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc).<br />Results: The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC&#95;2633 in a CCCTCTC motif-dependent manner.<br />Conclusion: We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.
- Subjects :
- Algorithms
Bacterial Proteins chemistry
Bacterial Proteins metabolism
Binding Sites
Gene Expression Regulation, Bacterial
High-Throughput Nucleotide Sequencing
Nucleotide Motifs
Oligonucleotides metabolism
Promoter Regions, Genetic
Protein Binding
Transcription Factors chemistry
User-Computer Interface
Computational Biology methods
DNA, Bacterial genetics
DNA, Bacterial metabolism
Transcription Factors metabolism
Xanthomonas campestris metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1471-2180
- Volume :
- 20
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- BMC microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 31896348
- Full Text :
- https://doi.org/10.1186/s12866-019-1672-7