Advances in genomic sequencing have allowed the identification of a multitude of genes encoding putative transcriptional regulatory proteins. Lacking, often, is a fuller understanding of the biological roles played by these proteins, the genes they regulate or regulon. Conventionally this is achieved through a genetic approach involving putative transcription factor gene manipulation and observations of changes in an organism’s transcriptome. However, such an approach is not always feasible or can yield misleading findings. Here, we describe a biochemistry-centric approach, involving identification of preferred DNA-binding sequences for the Thermus thermophilus HB8 transcriptional repressor TTHA0973 using the selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and bioinformatic analyses. We identified a consensus TTHA0973 recognition sequence of 5′–AACnAACGTTnGTT–3′ that exhibited nanomolar binding affinity. This sequence was mapped to several sites within the T. thermophilus HB8 genome, a subset of which corresponded to promoter regions regulating genes involved in phenylacetic acid degradation. These studies further demonstrate the utility of a biochemistry-centric approach for the facile identification of potential biological functions for orphan transcription factors in a variety of organisms.
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Identification of the Preferred DNA-Binding Sequence and Transcription Regulatory Network for the Thermophilic Zinc Uptake Regulator TTHA1292
ABSTRACT D-block metal cations are essential for most biological processes; however, excessive metal exposure can be deleterious to the survival of microorganisms. To tightly control heavy metal regulation, prokaryotic organisms have developed several mechanisms to sense and adapt to changes in intracellular and extracellular metal concentrations. The ferric uptake regulator superfamily of transcription factors associates with DNA when complexed with a regulatory metal cofactor and often represses the transcription of genes involved in metal transport, thus providing a genomic response to an environmental stressor. Although extensively studied in mesothermic organisms, there is little information describing ferric uptake regulator homologs in thermophiles. In this study, we biochemically characterize the ferric uptake regulator homolog TTHA1292 in the extreme thermophile Thermus thermophilus HB8. We identify the preferred DNA-binding sequence of TTHA1292 using the combinatorial approach, restriction endonuclease, protection, selection, and amplification (REPSA). We map this sequence to the Thermus thermophilus HB8 genome and identify the TTHA1292 transcription regulatory network, which includes the zinc ABC transporter subunit genes TTHA0596 and TTHA0453/4 . We formally implicate TTHA1292 as a zinc uptake regulator and show that zinc coordination is critical for the multimerization of TTHA1292 dimers on DNA in vitro and transcription repression in vivo . IMPORTANCE Discovering how organisms sense and adapt to their environments is paramount to understanding biology. Thermophilic organisms have adapted to survive at elevated temperatures (>50°C); however, our understanding of how these organisms adapt to changes in their environment is limited. In this study, we identify a zinc uptake regulator in the extreme thermophile Thermus thermophilus HB8 that provides a genomic response to fluctuations in zinc availability. These results provide insights into thermophile biology, as well as the zinc uptake regulator family of proteins.
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- NSF-PAR ID:
- 10413686
- Editor(s):
- Champion, Patricia A.
- Date Published:
- Journal Name:
- Journal of Bacteriology
- Volume:
- 204
- Issue:
- 11
- ISSN:
- 0021-9193
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Transcription factors are proteins that recognize specific DNA sequences and affect local transcriptional processes. They are the primary means by which all organisms control specific gene expression. Understanding which DNA sequences a particular transcription factor recognizes provides important clues into the set of genes that they regulate and, through this, their potential biological functions. Insights may be gained through homology searches and genetic means. However, these approaches can be misleading, especially when comparing distantly related organisms or in cases of complicated transcriptional regulation. In this work, we used a biochemistry-based approach to determine the spectrum of DNA sequences specifically bound by the Thermus thermophilus HB8 TetR-family transcription factor TTHB023. The consensus sequence 5′–(a/c)Y(g/t)A(A/C)YGryCR(g/t)T(c/a)R(g/t)–3′ was found to have a nanomolar binding affinity with TTHB023. Analyzing the T. thermophilus HB8 genome, several TTHB023 consensus binding sites were mapped to the promoters of genes involved in fatty acid biosynthesis. Notably, some of these were not identified previously through genetic approaches, ostensibly given their potential co-regulation by the Thermus thermophilus HB8 TetR-family transcriptional repressor TTHA0167. Our investigation provides additional evidence supporting the usefulness of a biochemistry-based approach for characterizing putative transcription factors, especially in the case of cooperative regulation.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/jb.00303-22
