Comparative Analysis of the IclR-Family of Bacterial Transcription Factors and Their DNA-Binding Motifs: Structure, Positioning, Co-Evolution, Regulon Content

Abstract

The IclR-family is a large group of transcription factors (TFs) regulating various biological processes in diverse bacteria. Using comparative genomics techniques, we have identified binding motifs of IclR-family TFs, reconstructed regulons and analyzed their content, finding co-occurrences between the regulated COGs (clusters of orthologous genes), useful for future functional characterizations of TFs and their regulated genes. We describe two main types of IclR-family motifs, similar in sequence but different in the arrangement of the half-sites (boxes), with GKTYCRYW_3-4RYGRAMC and TGRAACAN_1-2TGTTYCA consensuses, and also predict that TFs in 32 orthologous groups have binding sites comprised of three boxes with alternating direction, which implies two possible alternative modes of dimerization of TFs. We identified trends in site positioning relative to the translational gene start, and show that TFs in 94 orthologous groups bind tandem sites with 18-22 nucleotides between their centers. We predict protein-DNA contacts via the correlation analysis of nucleotides in binding sites and amino acids of the DNA-binding domain of TFs, and show that the majority of interacting positions and predicted contacts are similar for both types of motifs and conform well both to available experimental data and to general protein-DNA interaction trends.

Keywords: IclR-family; comparative genomics; protein-DNA contacts; tandem binding sites; transcription factor binding sites (TFBS); transcription regulation.

FIGURE 1

Example of a IclR-family three-box binding motif matching both group 1 and group 2 consensuses.

FIGURE 2

Joint LOGO diagrams of aligned binding sites of group 1 subgroups. Gaps are inserted to align even and odd binding sites.

FIGURE 3

Positioning of IclR-family binding sites. Horizontal axis – the distance between the site center and the gene start; vertical axis – the number of binding sites.

FIGURE 4

Distances between tandem binding sites. Horizontal axis – the distance between the adjacent site centers; vertical axis – the number of site pairs. Duplicates corresponding to divergently transcribed regulated genes are removed.

FIGURE 5

Heat map of correlations between amino acids and nucleotides for group 1 TFs and their binding sites. Sequence logos of HTH DNA-binding domains and their binding sites are shown on the top and to the left of the heat map, respectively. The total height of symbols at each position reflects the positional information content, whereas the height of individual symbols is proportional to the positional amino acid or nucleotide frequencies. The correlation scores are color-coded from yellow to red for amino acid-nucleotide pairs with statistical significance of correlation exceeding an automatically defined threshold (with red assigned for the most correlated pair). The violet-black palette is used for other pairs. Yellow lines denote positions where gaps have been removed from the amino acid alignment.

FIGURE 6

Heat map of correlations between amino acids and nucleotides for group 2 TFs and their binding sites. Notation as in Figure 5.

FIGURE 7

Joint LOGO diagrams of all group 1 and group 2 aligned binding sites. Arrows denote similar palindromic parts.

FIGURE 8

Amino acid positions involved in protein–DNA interaction of IclR-family TFs. TtgV in complex with DNA (PDB:2XRO) is chosen for visualization. Positions, for which protein–DNA correlations congruent with previous experimental and comparatively predicted data (see Table 5) are found, colored with red (even numbers) and brown (odd numbers).