Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973

Abstract

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.

Keywords: bioinformatics; biolayer interferometry (BLI); electrophoretic mobility shift assay (EMSA); extremophile; type IIS restriction endonuclease.

Figure 1

Selection and validation of TTHA0973-binding DNA sequences. (A) Shown are IR fluorescence images of restriction endonuclease cleavage protection assays made during Round 1 and Round 5 of REPSA selection with 17 nM TTHA0973 protein. The presence (+) or absence (−) of TTHA0973 and IISRE FokI (F) or BpmI (B) is indicated above each lane. The electrophoretic mobility of the intact (T) and cleaved (X) selection template, as well as the IRD7_ST2R primer (P), are indicated at the right of the figure. (B) Shown are IR fluorescence images of electrophoretic mobility shift assays made with DNA mixtures obtained following Round 1 (left lanes) and Round 5 (right lanes) of REPSA selection incubated with increasing concentrations of TTHA0973 protein (from left to right: 0, 0.8, 8, 80, or 800 nM TTHA0973). The electrophoretic mobility of a single protein-DNA complex (S) as well as uncomplexed ST2R24 selection template (T) and IRD7_ST2R primer (P) are indicated at the right of the figure.

Figure 2

TTHA0973 consensus sequences. Sequence logos were determined using MEME software with inputs of 1000 Round 5 DNA sequences. (A) MEME performed with no filters. (B) Palindromic filter.

Figure 3

EMSA analysis of TTHA0973-binding to consensus sequences. Shown are IR fluorescence images of IRD700-labeled TTHA0973 nonpalindromic (left) or palindromic (right), as indicated, incubated with 0, 10, 30, or 100 nM TTHA0973 protein. (S) Protein-DNA complex, (T) uncomplexed DNA, (P) IRD7_ST2R primer.

Figure 4

Biolayer interferometry analysis of TTHA0973 binding to DNA. Shown are raw traces (dots) and best-fit lines of TTHA0973 binding to (A) ST2_0973_R5_wt consensus DNA or (B) ST2_REPSAis control DNA. TTHA0973 concentrations investigated include 300 nM (red), 100 nM (green), 33 nM (blue), and 11 nM (magenta).

Figure 5

Bioinformatic identification of T. thermophilus HB8 promoters potentially regulated by TTHA0973. Shown are sequences +/− 200 bp of the TTHA0973-binding sequence identified through FIMO (see Table 2). Longest open reading frames with the same orientation as the target gene are indicated with blue nucleotides. Black nucleotides indicate intergenic regions; red nucleotides, overlapping open reading frames. Potential promoter elements (−30 and −10 boxes, +1 start site of transcription) identified using Softberry BPROM are indicated with cyan highlighting; those identified by U. Groningen PePPER by underlining. TTHA0973-binding sites are indicated with yellow highlighting. Overlapping TTHA0973-binding and core promoter elements are indicated by green highlighting.