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

Int J Mol Sci. 2021 Sep 17;22(18):10042. doi: 10.3390/ijms221810042.

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.

Keywords: bioinformatics; biolayer interferometry (BLI); electrophoretic mobility shift assay (EMSA); extremophile; protein-DNA binding; type IIS restriction endonuclease.

MeSH terms

  • Computational Biology
  • Electrophoretic Mobility Shift Assay
  • Extremophiles / metabolism
  • Gene Expression Regulation, Bacterial / genetics
  • Gene Expression Regulation, Bacterial / physiology
  • Thermus thermophilus / genetics
  • Thermus thermophilus / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Transcription Factors