REPSA: General combinatorial approach for identifying preferred ligand–DNA binding sequences

Dyke, Michael W. Van; Dyke, Natalya Van; Sunavala‐Dossabhoy, Gulshan

doi:10.1016/j.ymeth.2006.09.008

Cited by 11 publications

(6 citation statements)

References 17 publications

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“…Our SlmA structure, showing that it contains a N‐terminal HTH and is a TetR member, suggested that it may bind a palindromic DNA site as a homodimer, in a manner similar to other TetR proteins (Orth et al , 2000; Schumacher et al , 2002). With this a priori assumption, we went on to determine whether SlmA displays DNA‐binding specificity by conducting a restriction endonuclease protection, selection and amplification (REPSA) experiment (Van Dyke et al , 2007). The 43 unique possible binding sequences identified via REPSA were analysed with the sequence motif discovery program, Multiple Expectation Maximum for Motif Elicitation (MEME) (Supplementary Figure S4) (Bailey et al , 2006).…”

Section: Resultsmentioning

confidence: 99%

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check

et al. 2010

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Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in checkNucleoid occlusion (NO) restricts bacterial cell division to prevent chromosome guillotining in the cell midzone when replication or segregation is delayed. Structural work suggests that the NO factor SlmA (synthetic lethal with a defective Min system) interferes with formation of the cytokinetic Z-ring by altering associations between FtsZ protofilaments.

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Section: Resultsmentioning

confidence: 99%

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check

et al. 2010

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“…The other clones had varying regions of homology to their defined consensus sequence, ranging from three to 10 base pairs. While REPSA and genomic SELEX are related combinatorial selection methods, they have different strengths and weaknesses [17]. In this particular circumstance, genomic SELEX did not provide a substantially greater understanding of TTHA0973-DNA binding specificity than what was obtained through homology studies alone and was far inferior to the extent of data obtained by REPSA.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973

Cox¹,

Moncja²,

Mckinnes³

et al. 2019

IJMS

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Advances in genomic sequencing have allowed the identification of a multitude of genes encoding putative transcriptional regulatory proteins. Lacking, often, is a fuller understanding of the biological roles played by these proteins, the genes they regulate or regulon. Conventionally this is achieved through a genetic approach involving putative transcription factor gene manipulation and observations of changes in an organism’s transcriptome. However, such an approach is not always feasible or can yield misleading findings. Here, we describe a biochemistry-centric approach, involving identification of preferred DNA-binding sequences for the Thermus thermophilus HB8 transcriptional repressor TTHA0973 using the selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and bioinformatic analyses. We identified a consensus TTHA0973 recognition sequence of 5′–AACnAACGTTnGTT–3′ that exhibited nanomolar binding affinity. This sequence was mapped to several sites within the T. thermophilus HB8 genome, a subset of which corresponded to promoter regions regulating genes involved in phenylacetic acid degradation. These studies further demonstrate the utility of a biochemistry-centric approach for the facile identification of potential biological functions for orphan transcription factors in a variety of organisms.

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“…These libraries contained ST2R24 selection templates and 73-bp double-stranded deoxyribonucleotides with a central randomized 24-bp core flanked by defined sequences possessing IISRE-binding sites for FokI and BpmI [ 21 ]. Their design permitted the probing of sequence-specific protein-DNA binding through their inhibition of IISRE cleavage within the randomized core and the survival of intact templates for subsequent PCR amplification [ 37 ]. Defined, duplex DNA probes for biolayer interferometry (BLI) analyses were synthesized by PCR using ST2Ls and 5′-biotin-modified IRD7-ST2R primers, as previously described [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

Discovering the DNA-Binding Consensus of the Thermus thermophilus HB8 Transcriptional Regulator TTHA1359

Teague

Barrows

Baafi

et al. 2021

IJMS

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Transcription regulatory proteins, also known as transcription factors, function as molecular switches modulating the first step in gene expression, transcription initiation. Cyclic-AMP receptor proteins (CRPs) and fumarate and nitrate reduction regulators (FNRs) compose the CRP/FNR superfamily of transcription factors, regulating gene expression in response to a spectrum of stimuli. In the present work, a reverse-genetic methodology was applied to the study of TTHA1359, one of four CRP/FNR superfamily transcription factors in the model organism Thermus thermophilus HB8. Restriction Endonuclease Protection, Selection, and Amplification (REPSA) followed by next-generation sequencing techniques and bioinformatic motif discovery allowed identification of a DNA-binding consensus for TTHA1359, 5′–AWTGTRA(N)6TYACAWT–3′, which TTHA1359 binds to with high affinity. By bioinformatically mapping the consensus to the T. thermophilus HB8 genome, several potential regulatory TTHA1359-binding sites were identified and validated in vitro. The findings contribute to the knowledge of TTHA1359 regulatory activity within T. thermophilus HB8 and demonstrate the effectiveness of a reverse-genetic methodology in the study of putative transcription factors.

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REPSA: General combinatorial approach for identifying preferred ligand–DNA binding sequences

Cited by 11 publications

References 17 publications

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check

Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973

Discovering the DNA-Binding Consensus of the Thermus thermophilus HB8 Transcriptional Regulator TTHA1359

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