Molecular and structural considerations of TF-DNA binding for the generation of biologically meaningful and accurate phylogenetic footprinting analysis: the LysR-type transcriptional regulator family as a study model

Abstract

BackgroundThe goal of most programs developed to find transcription factor binding sites (TFBSs) is the identification of discrete sequence motifs that are significantly over-represented in a given set of sequences where a transcription factor (TF) is expected to bind. These programs assume that the nucleotide conservation of a specific motif is indicative of a selective pressure required for the recognition of a TF for its corresponding TFBS. Despite their extensive use, the accuracies reached with these programs remain low. In many cases, true TFBSs are excluded from the identification process, especially when they correspond to low-affinity but important binding sites of regulatory systems.ResultsWe developed a computational protocol based on molecular and structural criteria to perform biologically meaningful and accurate phylogenetic footprinting analyses. Our protocol considers fundamental aspects of the TF-DNA binding process, such as: i) the active homodimeric conformations of TFs that impose symmetric structures on the TFBSs, ii) the cooperative binding of TFs, iii) the effects of the presence or absence of co-inducers, iv) the proximity between two TFBSs or one TFBS and a promoter that leads to very long spurious motifs, v) the presence of AT-rich sequences not recognized by the TF but that are required for DNA flexibility, and vi) the dynamic order in which the different binding events take place to determine a regulatory response (i.e., activation or repression). In our protocol, the abovementioned criteria were used to analyze a profile of consensus motifs generated from canonical Phylogenetic Footprinting Analyses using a set of analysis windows of incremental sizes. To evaluate the performance of our protocol, we analyzed six members of the LysR-type TF family in Gammaproteobacteria.ConclusionsThe identification of TFBSs based exclusively on the significance of the over-representation of motifs in a set of sequences might lead to inaccurate results. The consideration of different molecular and structural properties of the regulatory systems benefits the identification of TFBSs and enables the development of elaborate, biologically meaningful and precise regulatory models that offer a more integrated view of the dynamics of the regulatory process of transcription.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3025-3) contains supplementary material, which is available to authorized users.

Deciphering the functional diversity of DNA-binding transcription factors in Bacteria and Archaea organisms

“…Diverse analysis has shown that genomic context can be useful to predict the function of genes regulated by TFs [ 41 ]. This approach is based on the observation that the genes coding for the TFs and target genes are often transcribed in opposite directions from overlapping or adjacent promoters, as occurs in the LysR family [ 42 ]; i.e., the transcriptional activation of the target gene occurs when one dimer of the TF binds to a binding site located adjacent the -35 box of the target gene promoter and interacts with the RNA polymerase. At the same time, the self-repression of the TF occurs when it binds to a TF-binding site that overlaps its own promoter located on the opposite strand of the DNA [ 42 ].…”

Functional Mechanism of the Efflux Pumps Transcription Regulators From Pseudomonas aeruginosa Based on 3D Structures

“…The family was named after the extensively studied transcription regulator of lysA implicated in lysine biosynthesis (Stragier et al, 1983 ) and is composed of both activators and repressors (Maddocks and Oyston, 2008 ) depending on the location of the transcription factor binding site (TFBS). The genetic organization of LTTRs targeted promoters and TFBSs has been studied by a computational protocol termed Phylogenetic Profile of Consensus Motifs issued by the analysis of Phylogenetic Footprinting technics (Oliver et al, 2016 ). In the LysR family, the gene coding for the TF is divergently oriented from its target gene (TG) and located <100 nucleotides upstream the beginning of the TG, and sometimes up to 500 bp from the initiation codon (Heroven and Dersch, 2006 ).…”

“…Depending on the affinity of the TF for one of the different TFBSs, the TF will be an activator or a repressor. The mean length of the TFBSs is 15 bp and the consensus sequence, originally described as 5'-T-n11-A-3' (Goethals et al, 1992 ), has been extended to 5′-CTATA-n9-TATAG-3′ (Oliver et al, 2016 ). Based on DNaseI protection assays combined with structural analysis (Wang and Winans, 1995 ; Muraoka et al, 2003 ; Picossi et al, 2007 ), a model has been proposed for the molecular mechanism of the LysR-type transcription regulators when three TFBSs are present, which is the majority of the LysR-type intergenic organization (Figure 4A ).…”

DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in the Thauera aromatica AR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway

“…The intergenic region includes a high-affinity binding site (primary binding site [PBS] [26], also known as the regulatory binding site [RBS]) that encompasses a T-N 11 -A conserved region showing certain dyad symmetry and a generally less-conserved secondary binding site (SBS; also known as the activating binding site [ABS]) partially overlapping the RNA polymerase Ϫ35 consensus sequence of the activated promoter. In many promoters, these two sequences show a certain degree of similarity (27). Once bound to the PBS and upon activation, e.g., through effector binding, the regulator undergoes a conformational change and establishes additional contacts with the SBS, which usually induces DNA bending, binding of the RNA polymerase, and transcription activation (24,28).…”