Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR

Abstract

One of the primary transcriptional regulators of fatty acid homeostasis in many prokaryotes is the protein FadR. To better understand its biological function in the extreme thermophile Thermus thermophilus HB8, we sought to first determine its preferred DNA-binding sequences in vitro using the combinatorial selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA) and then use this information to bioinformatically identify potential regulated genes. REPSA determined a consensus FadR-binding sequence , which was further characterized using quantitative electrophoretic mobility shift assays. With this information, a search of the T. thermophilus HB8 genome found multiple operons potentially regulated by FadR. Several of these were identified as encoding proteins involved in fatty acid biosynthesis and degradation; however, others were novel and not previously identified as targets of FadR. The role of FadR in regulating these genes was validated by physical and functional methods, as well as comparative genomic approaches to further characterize regulons in related organisms. Taken together, our study demonstrates that a systematic approach involving REPSA, biophysical characterization of protein-DNA binding, and bioinformatics can be used to postulate biological roles for potential transcriptional regulators.

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

“…Having identified TTHA0973-DNA consensus sequences by REPSA selection, we sought to validate these sequences through direct protein-DNA binding assays. EMSA, while qualitatively useful for determining different protein-DNA complexes, is not highly amenable for measuring kinetic binding parameters such as on- and off-rates [8]. Thus, we used BLI to determine the binding parameters of TTHA0973 to different DNAs.…”

“…Electrophoretic mobility shift assays (EMSA) with both libraries and defined DNAs were performed essentially as previously described [7,8], with a detailed protocol being available [19]. Biolayer interferometry was performed essentially as previously described [7], with the exception that only four concentrations of TTHA0973 (11, 33, 100, 300 nM) were used for each DNA probe investigated.…”

“…Our laboratory has developed an alternative, biochemistry-centric approach for determining the possible biological functions of putative transcription factors. First, we use the combinatorial selection method, Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and de novo motif discovery to determine a consensus DNA-binding sequence for a putative transcription factor [6,7,8]. This is then followed by motif scanning within the subject genome and bioinformatic analyses to identify potential regulated genes and their possible biological functions.…”

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

“…Electrophoretic mobility shift assays (EMSAs) with both Round 6 pool and TTHB023 consensus DNA were performed as previously described [4,5], with a detailed protocol being available [24]. Binding reactions (5 µL) consisted of 1 ng (21 fmoles) DNA and indicted concentration of TTHB023 in 1 × NEB CutSmart buffer.…”

“…We have developed an alternative, biochemistry-based approach, using the selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA), massively parallel sequencing, and bioinformatics to determine a consensus binding sequence and thereby identify possible genes regulated by these transcription factors. We have successfully used this approach to investigate three putative TetR-family transcription factors, TTHA0167, TTHA0101, and TTHA0973, in the extreme thermophile Thermus thermophilus HB8 [4][5][6]. Here, we describe our investigations into the DNA-binding specificity and genomic targets of TTHB023, a putative TetR-related transcriptional repressor protein in T. thermophilus HB8.…”