General and Genomic DNA-Binding Specificity for the Thermus thermophilus HB8 Transcription Factor TTHB023

Abstract

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.

Determination and Dissection of DNA-Binding Specificity for the Thermus thermophilus HB8 Transcriptional Regulator TTHB099

“…TTHB099 protein was expressed following IPTG induction of E. coli BL21(DE3) bacteria transformed with plasmid PC014099-42 (obtained from RIKEN Bioresource Research Center) and purified from soluble bacterial extracts by heat-treatment as described in our previous study [11]. SDS-PAGE analysis of fractions from purification steps is shown in Supplementary Figure S2A and is consistent with a near quantitative recovery of TTHB099 protein.…”

“…REPSA was used to select for TTHB099-binding sites present in a large pool (~60 billion molecules) of synthesized double-stranded DNA. Our selection library, ST2R24, has been successfully used in four previous studies [8][9][10][11]. IRDye ® 700 (IRD7)-labeled library DNA was incubated with purified TTHB099 protein to permit specific binding, then challenged by a type IIS restriction endonuclease (IISRE).…”

“…Note that EMSA experiments performed with REPSA selected DNAs contain multiple DNA species, including high concentrations of DNA primers, and should not be used to determine apparent binding affinities. Biolayer interferometry was performed as previously described [11], with the exception that only four concentrations of TTHB099 (17, 50, 150, 450 nM) were used for each DNA probe investigated. Such was sufficient to yield global values for k on and k off rate constants as well as K D equilibrium binding constants with R 2 goodness-of-fit determinations of greater than 0.95 in all cases.…”

“…To gain insights into transcriptional regulatory networks in extremophile organisms, our laboratory has employed a novel biochemistry-based method, Restriction Endonuclease, Selection, Protection, and Amplification (REPSA), to characterize several TFs in the extreme thermophilic model organism Thermus thermophilus HB8. To date, we have studied four tetracycline repressor protein (TetR) family transcriptional suppressors and have successfully identified their TFBSs [8][9][10][11]. Commonly, suppressors bind DNA in the absence of small-molecule modulators/cofactors and with high-affinity.…”

Biomolecules from Different Angles

“…Therefore, this Special Issue is a snapshot of a collective view of Biomolecules on biomolecules. The covered topics range from the description of secondary metabolites produced by fungi [1] to the overview of the phytochemical composition and pharmacological and food applications of Prosopis plants [2], to comparative evaluation of the antiproliferative activity of plant-derived silver nanoparticles synthesized using callus and anthocyanin extracts of purple basil (BC-AgNPs and AE-AgNPs, respectively) [3], to systematic description of the utilization of natural products as a source of antitumor drugs [4], to the analysis of the effects of caffeine and other methylxanthines on Aβ-homeostasis by shifting the αand β-secretase-based processing of amyloid precursor protein (APP) from the Aβ-producing amyloidogenic to the non-amyloidogenic pathway [5], to the topoisomerase I and radical trapping antioxidant activities of a series of N-alkyl-acridones and N,N -dialkyl-9,9 -biacridylidenes [6], to the analysis of the effects of phytoalexins in soybeans via synergistic inhibition on α-glucosidase [7], to functional and structural characterization of several important proteins (such as the analysis of the general and genomic DNA-binding specificity of a transcription factor TTHB023 from Thermus thermophilus HB8 [8], investigation of the voltage sensing in protein translocation via the bacterial channel SecYEG [9], and structural characterization of the terminase (packaging protein) pUL15, which is the most conserved among all the Herpes Simplex Virus 1 (HSV1) gene products [10]), to the description of utilization of kinetic transition in amyloid assembly for screening fluorophores for preferential responses to oligomer over fibril formation [11], to the description of the utilization of amyloid fibril biomaterials, designer amyloid cellpenetrating peptides comprised of β-sheet cores derived from naturally occurring protein sequences and designed positively charged and aromatic residues exposed at key residue positions as gene transfer vehicles capable of self-assembling and promoting the DNA condensation and cell internalization [12], to the introduction of the use of phage display system for generation of lamprey monoclonal antibodies, lampribodies, which are the variable lymphocyte receptors (VLRs) consisting of leucine rich repeats (LRRs), whose diversity is generated by stepwise genomic rearrangements of LRR cassettes dispersed throughout the VLRB locus [13], to the description of changes in the N-glycomic profile associated with post-traumatic stress disorder (PTSD) via comparative analysis of the N-glycomic profiles in 543 male Caucasian individuals (299 veterans with PTSD and 244 control subjects) [14], to the analysis of the effect of macromolecular crowding on the functionality of tumor suppressors ING containing a plant homeodomain (PHD) and their ability to interact with the histone H3 trimethylated at lysine 4 (H3K4me3) [15], and to the comparison of...…”