Deciphering the protein‐DNA code of bacterial winged helix‐turn‐helix transcription factors

Abstract

Background: Sequence-specific binding by transcription factors (TFs) plays a significant role in the selection and regulation of target genes. At the protein:DNA interface, amino acid side-chains construct a diverse physicochemical network of specific and non-specific interactions, and seemingly subtle changes in amino acid identity at certain positions may dramatically impact TF:DNA binding. Variation of these specificity-determining residues (SDRs) is a major mechanism of functional divergence between TFs with strong structural or sequence homology. Methods: In this study, we employed a combination of high-throughput specificity profiling by SELEX and Spec-seq, structural modeling, and evolutionary analysis to probe the binding preferences of winged helix-turn-helix TFs belonging to the OmpR sub-family in Escherichia coli. Results: We found that E. coli OmpR paralogs recognize tandem, variably spaced repeats composed of "GT-A" or "GCT"-containing half-sites. Some divergent sequence preferences observed within the "GT-A" mode correlate with amino acid similarity; conversely, "GCT"-based motifs were observed for a subset of paralogs with low sequence homology. Direct specificity profiling of a subset of OmpR homologues (CpxR, RstA, and OmpR) as well as predicted "SDR-swap" variants revealed that individual SDRs may impact sequence preferences locally through direct contact with DNA bases or distally via the DNA backbone. Conclusions: Overall, our work provides evidence for a common structural code for sequence-specific wHTH:DNA interactions, and demonstrates that surprisingly modest residue changes can enable recognition of highly divergent sequence motifs. Further examination of SDR predictions will likely reveal additional mechanisms controlling the evolutionary divergence of this important class of transcriptional regulators.Keywords: transcription factor; SELEX; winged helix-turn-helix; specificity determinants; two-component signaling Author summary: Although many transcription factors (TFs) possess high sequence similarity, subtle amino acid variation at DNA-contacting positions can yield substantial (and difficult to predict) alterations to intrinsic recognition potential. In this work, we characterized the natural variation in recognition potential (base preference, monomer spacing, and monomer orientation) within a sub-family of E. coli winged helix-turn-helix TFs. Using patterns of amino acid conservation, we further predicted a number of amino acids with likely involvement in specificity determination between these related TFs. Finally, we demonstrated the complex local and global roles of predicted SDRs as well as protein sequence context on sequence-specific binding.

Exploring the mono-/bistability range of positively autoregulated signaling systems in the presence of competing transcription factor binding sites

“…The fluorescent reporter was integrated into the HK022 phage attachment site in the chromosome of BW25113 using recombination strategies [ 64 ], yielding RU2118. Decoy CusR binding sites were made by annealing oligonucleotides (see details in S1 Text ) with sequences based on previous analyses [ 34 , 65 ] and inserted into pRG475 [ 18 ], a plasmid with a copy number that has been determined previously and that can be further manipulated by arabinose. The resulting plasmids pCusRBS1 (1 site/plasmid), pCusRBS2 (2 sites), pCusRBS3 (3 sites) and pLH10 (0 site) [ 18 ] were introduced into RU2118 for flow cytometry analyses.…”