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.

Graphical abstract

Keywords

transcription factor / SELEX / winged helix-turn-helix / specificity determinants / two-component signaling

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SUPPLEMENTARY MATERIALS

The supplementary materials can be found online with this article at DOI 10.1007/s40484-018-0130-0.

ACKNOWLEDGEMENTS

We are grateful for the thoughtful input of former members of the laboratory Benjamin Borgo and Chi Zhang in the initial phases of this work, and for continuing helpful discussions and whose technical expertise greatly added to this work. We thank Dr. Cailin Joyce and Dr. GiNell Elliott for their critical commentary during the preparation of this manuscript. We especially thank Jessica Hoisington-Lopez for her input and heroic patience in our SELEX library design and the development of sequencing approaches. This project was completed with support of NSF Graduate Research Fellowship Award DGE-1143954 (to A.P.J.) and National Institutes of Health Award Number R01GM101602 (to J.J.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors report no potential conflicts of interest.

COMPLIANCE WITH ETHICS GUIDELINES

The authors Adam P. Joyce and James J. Havranek declare that they have no conflict of interests.ƒThis article does not contain any studies with human or animal subjects performed by any of the authors.

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