Discovering the DNA-Binding Consensus of the Thermus thermophilus HB8 Transcriptional Regulator TTHA1359

Abstract

Transcription regulatory proteins, also known as transcription factors, function as molecular switches modulating the first step in gene expression, transcription initiation. Cyclic-AMP receptor proteins (CRPs) and fumarate and nitrate reduction regulators (FNRs) compose the CRP/FNR superfamily of transcription factors, regulating gene expression in response to a spectrum of stimuli. In the present work, a reverse-genetic methodology was applied to the study of TTHA1359, one of four CRP/FNR superfamily transcription factors in the model organism Thermus thermophilus HB8. Restriction Endonuclease Protection, Selection, and Amplification (REPSA) followed by next-generation sequencing techniques and bioinformatic motif discovery allowed identification of a DNA-binding consensus for TTHA1359, 5′–AWTGTRA(N)6TYACAWT–3′, which TTHA1359 binds to with high affinity. By bioinformatically mapping the consensus to the T. thermophilus HB8 genome, several potential regulatory TTHA1359-binding sites were identified and validated in vitro. The findings contribute to the knowledge of TTHA1359 regulatory activity within T. thermophilus HB8 and demonstrate the effectiveness of a reverse-genetic methodology in the study of putative transcription factors.

Capture-SELEX: Selection Strategy, Aptamer Identification, and Biosensing Application

“…Optical-fiber-based BLI biosensors have been developed for real-time, sensitive, and rapid measurement [ 112 ] of interactions between biomolecules, including DNA–protein [ 113 , 114 ], antibody–antigen [ 115 ], and DNA–small-molecule-contaminant interactions [ 116 , 117 ]. The sensor tip of an optical fiber consists of two reflective surfaces: a streptavidin-coated biocompatible surface, which is immersed in the sample solution, and an optical layer, which functions as an internal surface ( Figure 3 A).…”

Guidelines on the performance evaluation of motif recognition methods in bioinformatics

“…The accurate discovery of DNA and RNA regulatory motifs and their combinations is still a topic of active research, focusing to date mainly on the analysis of ChIP-Seq data ( Kingsley et al, 2019 ; Kong et al, 2020 ), on gene co-expression analysis ( Rouault et al, 2014 ; Teague et al, 2021 ) and on the general investigation of the properties of binding motifs ( Zeitlinger 2020 ). Many bioinformatics methods have been ( Zambelli et al, 2013 ) and are still being developed ( Bentsen et al, 2022 ; Hammelman et al, 2022 ) to improve prediction accuracy and fully address the advantages of novel experimental and computational techniques, such as that based on deep learning ( Auslander et al, 2021 ).…”