Student or educator? Sign up with your school email and get 6 months of Pro free.
CoChat
Sign Up
Cited by 3Bacterial Genetics and BiotechnologyGenomics and Phylogenetic StudiesGenomics and Chromatin Dynamics

The DOI matches its Crossref record; 45 of the 45 references it cites were found in a registry or an index.

The Evolution of Function in the DNA-Binding Domain of the CRP/FNR Family

Open access
Krishnaswamy, Madhumitha, Seshasayee, Aswin Sai Narain·2025·Genome Biology and Evolution
DOI 10.1093/gbe/evaf182PMC PMC12531629

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

Transcriptional regulation, facilitated by transcription factors (TFs), contributes to bacterial response to environmental and cellular perturbations. How have TFs diversified and gained their functions? We use the cyclic AMP receptor protein (CRP)/regulator of fumarate and nitrate reductase (FNR) family to study this question across ∼6,000 prokaryotic genomes. Characterized by homology to TFs CRP and FNR in Escherichia coli, the two functions of this family—sequence-specific DNA binding and transcriptional activation by direct contacts to RNA polymerase—are conferred by the helix–turn–helix containing DNA-binding domain. We constructed a rooted phylogeny of this domain and performed residue conservation analysis on extant and in silico reconstructed ancestral sequences of CRP/FNR family members. The overall ancestor of the sequence family does not fall into classes containing either E. coli CRP (Eco-CRP) or E. coli FNR (Eco-FNR) clades, but instead a large and diverse class that we call “CRP-like.” Residues key for base-specific DNA binding remain largely conserved in the family, and likely emerged in the family common ancestor. Residues (together called activating region 1 site [AR1]) that make direct contacts with α-C-terminal domain (αCTD) of RNA polymerase are conserved only in the Eco-CRP class and restricted to Gammaproteobacteria. The corresponding “287 determinant” interface on αCTD is again fully conserved only in Gammaproteobacteria, which contain Eco-CRP members, following stepwise evolution initiated at the Proteobacterial common ancestor. These suggest suggesting co-emergence of the interface residues in Eco-CRP. Our analysis hence shows that class I transcriptional activation through AR1 on CRP and the determinant of αCTD, as described in E. coli CRP, is phylogenetically restricted, while base-specific DNA binding has been present in the CRP/FNR family throughout its evolutionary history.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1093/gbe/evaf182

Cites

45 of 45 references verified0 citation errors · 0 not checkable

Statistics

Figures

Fig. 1. Classification and phylogeny of the c-f-HTH domain. a) Heatmap showing the presence/absence (black/white) of the different classes across all taxa which showed the presence of the c-f-HTH domain (N = 4,114), represented at the phylum level (class level for certain diverse phyla). Note: grayscale heatmap with thin binary values. b) Collapsed gene tree (N = 10,224) of the c-f-HTH domain sequences rooted using the DNA-binding domain of a methyltransferase enzyme from Fungi. The size of collapsed nodes indicates the number of extant tips, and colored based on classification. Collapsed and visualized using iTOL. c) Collapsed Neighbor Joining (NJ) tree (N = 10,227) of c-f-HTH domain sequences along with four key ancestral nodes, as indicated in legend. Clades containing nodes of interest are shaded on the basis of overall regulators present in the clade. Collapsed and visualized using iTOL.
Fig. 1. Classification and phylogeny of the c-f-HTH domain. a) Heatmap showing the presence/absence (black/white) of the different classes across all taxa which showed the presence of the c-f-HTH domain (N = 4,114), represented at the phylum level (class level for certain diverse phyla). Note: grayscale heatmap with thin binary values. b) Collapsed gene tree (N = 10,224) of the c-f-HTH domain sequences rooted using the DNA-binding domain of a methyltransferase enzyme from Fungi. The size of collapsed nodes indicates the number of extant tips, and colored based on classification. Collapsed and visualized using iTOL. c) Collapsed Neighbor Joining (NJ) tree (N = 10,227) of c-f-HTH domain sequences along with four key ancestral nodes, as indicated in legend. Clades containing nodes of interest are shaded on the basis of overall regulators present in the clade. Collapsed and visualized using iTOL.CC BY · PubMed Central
Fig. 2. Conservation of key functional residues. Heatmaps for each key residue of interest across the different domain classes, ordered by physicochemical properties. Color of intersecting boxes is scaled according to the amino acid frequency from low (light) to high (dark) and indicates the occurrence of a particular amino acid for the specified key residue in all members of a class. The distributions for Eco-CRP are indicated in the green dashed boxes.
Fig. 2. Conservation of key functional residues. Heatmaps for each key residue of interest across the different domain classes, ordered by physicochemical properties. Color of intersecting boxes is scaled according to the amino acid frequency from low (light) to high (dark) and indicates the occurrence of a particular amino acid for the specified key residue in all members of a class. The distributions for Eco-CRP are indicated in the green dashed boxes.CC BY · PubMed Central
Fig. 3. Conservation and emergence in the gene tree. Collapsed outgroup rooted gene trees of the c-f-HTH domain (N = 10,224) colored on a grayscale gradient (low to high) on the basis of PSSM scores calculated for a) activating region 1 (AR1) residues, b) Residues involved in making phosphate backbone contacts, c) Residues involved in making specific base contacts with DNA. The size of collapsed nodes corresponds to the number of extant tips. Legend in gray box is Fig. 1b replicated for reference to identity of different clades in the gene tree. Collapsed and visualized using iTOL.
Fig. 3. Conservation and emergence in the gene tree. Collapsed outgroup rooted gene trees of the c-f-HTH domain (N = 10,224) colored on a grayscale gradient (low to high) on the basis of PSSM scores calculated for a) activating region 1 (AR1) residues, b) Residues involved in making phosphate backbone contacts, c) Residues involved in making specific base contacts with DNA. The size of collapsed nodes corresponds to the number of extant tips. Legend in gray box is Fig. 1b replicated for reference to identity of different clades in the gene tree. Collapsed and visualized using iTOL.CC BY · PubMed Central
Fig. 4. Patterns of conservation of αCTD. a) Smoothscatter plot and scatterplot of PSSM scores corresponding to 287 determinants of αCTD versus AR1 scores of Eco-CRP and CRP-like regulators. The density of the points is shown in the smooth scatter plot while the points are colored on the basis of class in the scatter plot. Regression lines are plotted in gray for CRP-like and Eco-CRP. b) Conservation of the 287 determinant mapped onto the 16S rRNA species tree (N = 1,454). PSSM scores of the 287 determinants divided into four categories as indicated in legend—extremely low, low, medium, and high, mapped onto their corresponding genomes on the species tree. Colourstrip annotations show key classes present and the taxonomic annotation of the genome. Note, for simplicity, extremely low and low categories are combined into one in the text. Colored and visualized using iTOL.
Fig. 4. Patterns of conservation of αCTD. a) Smoothscatter plot and scatterplot of PSSM scores corresponding to 287 determinants of αCTD versus AR1 scores of Eco-CRP and CRP-like regulators. The density of the points is shown in the smooth scatter plot while the points are colored on the basis of class in the scatter plot. Regression lines are plotted in gray for CRP-like and Eco-CRP. b) Conservation of the 287 determinant mapped onto the 16S rRNA species tree (N = 1,454). PSSM scores of the 287 determinants divided into four categories as indicated in legend—extremely low, low, medium, and high, mapped onto their corresponding genomes on the species tree. Colourstrip annotations show key classes present and the taxonomic annotation of the genome. Note, for simplicity, extremely low and low categories are combined into one in the text. Colored and visualized using iTOL.CC BY · PubMed Central
Fig. 5. The evolutionary trajectory of functions in the c-f-HTH domain of the CRP/FNR family. Conservation of the key residues identified in the study marked the 16S rRNA species tree. Colorstrip annotations (innermost to outermost) correspond to the range in PSSM scores observed for the 287 determinant, AR1, and specific base contact residues. For genomes coding for multiple CRP/FNR family proteins, AR1 and specific base contact scores for the best-scoring protein are shown. Branches are colored on the basis of taxonomic annotation, and Gammaproteobacteria, showing the highest scores for 287 determinants of αCTD, along with AR1 and specific base contact scores, is highlighted. Colored and visualized using iTOL.
Fig. 5. The evolutionary trajectory of functions in the c-f-HTH domain of the CRP/FNR family. Conservation of the key residues identified in the study marked the 16S rRNA species tree. Colorstrip annotations (innermost to outermost) correspond to the range in PSSM scores observed for the 287 determinant, AR1, and specific base contact residues. For genomes coding for multiple CRP/FNR family proteins, AR1 and specific base contact scores for the best-scoring protein are shown. Branches are colored on the basis of taxonomic annotation, and Gammaproteobacteria, showing the highest scores for 287 determinants of αCTD, along with AR1 and specific base contact scores, is highlighted. Colored and visualized using iTOL.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Metadata

Authors
Krishnaswamy, M., & Seshasayee, A. S. N.
Affiliations
Tata Institute of Fundamental Research (IN)
Corresponding author
Seshasayee, Aswin Sai Narain
ORCID
Venue
Genome Biology and Evolution
Publisher
Oxford University Press (OUP)
Published
2025-09-25
Volume
17
Issue
10
ISSN
1759-6653 (ISSN-L)
DOI
10.1093/gbe/evaf182
PMC
PMC12531629
OpenAlex
W4414500780
Cited by
3
References
45
Citations by year
2026: 3
FWCI
1.75
Type
journal article
Language
en
Open access
Licence
Locations
  • Genome Biology and Evolution (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Department of Atomic Energy, Government of India (RTI 4006)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
transcription factors, DNA-binding domain, helix-turn-helix, transcriptional activation, ancestral sequence reconstruction, protein evolution, Gammaproteobacteria, bacterial transcriptional regulation, phylogenetic analysis, amino acid conservation
MeSH terms
Protein Domains, Cyclic AMP Receptor Protein, DNA-Binding Proteins, Escherichia coli, Iron-Sulfur Proteins, Phylogeny, Prokaryotic Cells, Sporothrix, Transcription Factors, Evolution, Molecular

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

Data sources

Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.