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

International Journal of Molecular Sciences

Cynthia A. Baafi¹  Michael W. Van Dyke¹  Josiah L. Teague¹  John K. Barrows¹ 

[1] Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, GA 30144, USA;

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

DOI  :  10.3390/ijms221810042

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown