Structural basis of direct and inverted DNA sequence repeat recognition by helix–turn–helix transcription factors

Fernández-López, Raúl; Ruiz, Raúl; Campo, Irene del; González-Montes, Lorena; Boer, D.R.; Cruz, Fernando de la; Moncalián, Gabriel

doi:10.1093/nar/gkac1024

Cited by 6 publications

(3 citation statements)

References 33 publications

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“…Based on sequence analysis, this upstream region contains plausible - 35 and -10 promoter sites as well as an inverted repeat motif (Figure S2). The observed point mutation occurs in the predicted inverted repeat motif and could potentially interfere with binding of a transcriptional regulator (34). Repeated attempts to reconstruct this single nucleotide mutation in a wild-type background were unsuccessful.…”

Section: Resultsmentioning

confidence: 99%

Laboratory evolution inNovosphingobium aromaticivoransenables rapid catabolism of a model lignin-derived aromatic dimer

Allemann,

Kato,

Alexander

et al. 2024

Preprint

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Lignin contains a variety of interunit linkages, which leads to a range of potential decomposition products that can be used as carbon sources by microbes. β-O-4 linkages are the most common in native lignin and associated catabolic pathways have been well characterized. However, the fate of the mono-aromatic intermediates that result from β-O-4 dimer cleavage has not been fully elucidated. Here, we used experimental evolution to identify mutant strains of Novosphingobium aromaticivorans with improved catabolism of a model aromatic dimer containing a β-O-4 linkage, guaiacylglycerol-β-guaiacyl ether (GGE). We identified several parallel causal mutations, including a single nucleotide mutation in the promoter of an uncharacterized gene that roughly doubled the growth yield with GGE. We characterized the associated enzyme and demonstrated that it oxidizes an intermediate in GGE catabolism, β-hydroxypropiovanillone, to vanilloyl acetaldehyde. Identification of this enzyme and its key role in GGE catabolism furthers our understanding of catabolic pathways for lignin-derived aromatic compounds.

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Section: Resultsmentioning

confidence: 99%

Laboratory evolution inNovosphingobium aromaticivoransenables rapid catabolism of a model lignin-derived aromatic dimer

Allemann,

Kato,

Alexander

et al. 2024

Preprint

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“…This analysis revealed a coarse correlation (Supplementary Figure 7) suggesting that if the known operator is found in the upstream, inter-operon region, then the chances of it being correctly predicted increase. To further enhance performance the search algorithm can potentially be improved by adding the option to search for direct repeats rather than inverted repeats 36 . Moreover, as the number of validated operators expands, "seed sequence motifs" can be used as queries, potentially avoiding the identification of false positives in the form of terminators or binding sequences of other DNA-binding proteins.…”

Section: Discussionmentioning

confidence: 99%

Snowprint: a predictive tool for genetic biosensor discovery

d’Oelsnitz

Stofel

Ellington

2023

Preprint

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Bioengineers increasingly rely on ligand-inducible transcription regulators for chemical-responsive control of gene expression, yet the number of regulators available is limited. Novel regulators can be mined from genomes, but an inadequate understanding of their DNA specificity complicates genetic design. Here we present Snowprint, a simple yet powerful bioinformatic tool for predicting regulator:DNA interactions. Benchmarking results demonstrate that Snowprint predictions are significantly similar for >45% of experimentally validated regulator:operator pairs from organisms across nine phyla and for regulators that span five distinct structural families. We then use Snowprint to design promoters for 33 previously uncharacterized regulators sourced from diverse phylogenies, of which 28 were shown to influence gene expression and 24 produced a >20-fold signal-to-noise ratio. A panel of the newly domesticated regulators were then screened for response to biomanufacturing-relevant compounds, yielding new sensors for a polyketide (olivetolic acid), terpene (geraniol), steroid (ursodiol), and alkaloid (tetrahydropapaverine) with induction ranges up to 10.7-fold. Snowprint represents a unique, generalizable tool that greatly facilitates the discovery of ligand-inducible transcriptional regulators for bioengineering applications.

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“…The basic HTH domain consists of three core helices making up a right-handed helical bundle with a partly open configuration, where typically two HTH domains are used to recognise the operator containing inverted or direct repeat sequence elements. The efficiency of binding of a repressor protein to an operator site depends on its binding affinity, which again is determined by base-specific readouts and intrinsic DNA shape, however, the exact mechanism by with repressor proteins specifically recognise their DNA binding site(s) in vivo remains unclear (7)(8)(9).…”

Section: Introductionmentioning

confidence: 99%

Asymmetry of both repressor and operator is important for the transcriptional regulation of theP. putidaXre-RES system

Henriksen,

Brodersen,

Skjerning

2024

Preprint

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Transcriptional regulation is a fundamental mechanism in bacteria and is often mediated by repressor proteins. In the type II toxin-antitoxin (TA) system. xre-res, the Xre antitoxin contains a putative helix-turn-helix (HTH) DNA-binding domain and could thus potentially bind to and repress transcription from the xre-res promoter. The structure of the Pseudomonas putida Xre-RES TA complex revealed an unusual 4:2 stoichiometry with two potential DNA-binding sites, suggesting a non-canonical mechanism of transcriptional autoregulation. Here, we show that the activity of the xre-res promoter requires both an intact sigma70 element and the transcriptional start site, and that the Xre-RES complex represses transcription via binding to an imperfect inverted repeat region downstream of the sigma70 element. We furthermore confirm the presence of the unusual 4:2 TA complex in solution and show that it preferably binds the imperfect inverted repeat in a 1:1 ratio. In addition, we show that the isolated Xre antitoxin is an aggregation-prone monomer in vitro and a weak repressor in vivo. We find that the Xre dimer can dissociates within the 4:2 complex and result in a 2:2 TA complex that still neutralizes the RES toxin but cannot bind DNA. Together, our data suggests that the asymmetry of the promoter is important for both transcription and repression, and we propose a model in which the Xre-RES complex regulates transcription through a dynamic and concentration-dependent equilibrium between a non-binding (2:2) and a DNA-binding (4:2) form.

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Structural basis of direct and inverted DNA sequence repeat recognition by helix–turn–helix transcription factors

Cited by 6 publications

References 33 publications

Laboratory evolution inNovosphingobium aromaticivoransenables rapid catabolism of a model lignin-derived aromatic dimer

Laboratory evolution inNovosphingobium aromaticivoransenables rapid catabolism of a model lignin-derived aromatic dimer

Snowprint: a predictive tool for genetic biosensor discovery

Asymmetry of both repressor and operator is important for the transcriptional regulation of theP. putidaXre-RES system

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