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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. 

Our laboratory studies putative transcription regulatory proteins from the extreme thermophile, Thermus thermophilus HB8. To do so, these proteins are often expressed in the mesothermic organism Escherichia coli and purified from whole-cell extracts. Here we describe our standard expression and purification scheme and its analysis using the thermostable protein His6-GFP-TEV. His6-GFP-TEV is usually used as an N-terminus tag for the bacterial expression of recombinant proteins. However, this surrogate provides a facile visual indicator of thermostable protein expression and purification and is especially amenable for laboratory instruction in a primarily undergraduate educational environment. 

Transcription factors (TFs) have been extensively researched in certain well-studied organisms, but far less so in others. Following the whole-genome sequencing of a new organism, TFs are typically identified through their homology with related proteins in other organisms. However, recent findings demonstrate that structurally similar TFs from distantly related bacteria are not usually evolutionary orthologs. Here we explore TTHB099, a cAMP receptor protein (CRP)-family TF from the extremophile Thermus thermophilus HB8. Using the in vitro iterative selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), we identified the preferred DNA-binding motif for TTHB099, 5′–TGT(A/g)NBSYRSVN(T/c)ACA–3′, and mapped potential binding sites and regulated genes within the T. thermophilus HB8 genome. Comparisons with expression profile data in TTHB099-deficient and wild type strains suggested that, unlike E. coli CRP (CRPEc), TTHB099 does not have a simple regulatory mechanism. However, we hypothesize that TTHB099 can be a dual-regulator similar to CRPEc. 

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. 

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. 

Short DNA probes, for example, those used in characterizing protein-DNA complexes, have historically been radiolabeled with 32P to allow their detection and quantitation following native polyacrylamide gel electrophoresis (PAGE). For reasons of economy or safety, alternative means of DNA detection need to be used. Described here is the resolution of 5' IRD700 and IRD800 end-labeled DNAs by native PAGE and their visualization and quantitation by IR fluorescence imaging. 

R fluorophore-modified DNAs and IR fluorescence imaging provide a safe and effective alternative for investigating ligand-DNA interactions than those involving radioactivity. These DNAs can be synthesized by conventional phosphoramidite chemistries and are commercially available. However, they are relatively expensive, which can be prohibitive if many different DNA probes are required. Described here is the design of modular DNA probes that can be synthesized by PCR using conventional templates and a defined set of IR fluorophore-modified primers. These have been found effective for investigating protein-DNA interactions in a variety of assays, including Electrophoretic Mobility Shift Assays (EMSA), Restriction Endonuclease Protection Assays (REPA), and the iterative selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA). 

Protein-DNA binding interactions are critical in several biological processes, especially the regulation of gene expression at the level of transcription initiation. An important technique for studying these interactions is the electrophoretic mobility shift assay (EMSA), whereby protein-DNA complexes are resolved on the basis of their mass:charge ratio using native polyacrylamide gel electrophoresis (nPAGE). Here we describe EMSA using PCR-generated, near infrared-fluorescent DNA probes, and IR fluorescence imaging to qualitatively and quantitatively study the interaction of transcriptional regulatory proteins from thermophilic organisms with different DNAs. Direct imaging of IR fluorophore-labeled DNA probes is advantageous because it provides high sensitivity (subnanomolar) without the need for intermediate staining steps or costly and problematic radiolabeled probes, thereby providing a more affordable and sensitive option to image protein-DNA on polyacrylamide gels by techniques such as EMSA. 

For convenience and for PCR products that are challenging to purify with high efficiency (e.g., chemically modified DNAs), it is often desirable to quantitate synthesized DNA directly from a PCR reaction. Here we describe the use of a high-sensitivity Quant-iT™ PicoGreen® dye-based fluorescence assay to quantitate PCR-synthesized, double-stranded, low molecular weight, 5’-modified DNA probes in the presence of single-stranded primers and deoxyribonucleotides.