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.
Highlights
In bacteria, transcription regulatory proteins or transcription factors function as critical constituents of signal transduction networks, acting upon environmental and cellular cues to modulate the transcriptional program appropriately through their specific binding to control elements within targeted gene promoters [1]
While their functions are traditionally determined through genetic means, this is less feasible in many less-well-studied organisms, often relying on genomic organization and structural homology to infer putative transcription factor biological roles [2]
Following complexation with the metabolite effector 30 −50 cAMP, CRPEc homodimers adopt a conformation that allows them to bind to DNA sequences with the consensus 50 -AAATGTGAtctagaTCACATTT-30, thereby regulating hundreds of genes involved with the catabolism of secondary carbon sources [6,7,8]
Summary
Transcription regulatory proteins or transcription factors function as critical constituents of signal transduction networks, acting upon environmental and cellular cues to modulate the transcriptional program appropriately through their specific binding to control elements within targeted gene promoters [1]. While their functions are traditionally determined through genetic means, this is less feasible in many less-well-studied organisms, often relying on genomic organization and structural homology to infer putative transcription factor biological roles [2]. FNREc , on the other hand, forms homodimers containing two [4Fe-4S]
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