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 5´-TTRNACYNRGTNYAA-3´, 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.
Highlights
Genome projects have yielded considerable information since the sequencing of the first whole microorganism genome, Haemophilus influenza, in 1995 [1,2]
We describe the application of REPSA to determine the preferred DNA-binding sequences for the T. thermophilus HB8 transcription factor FadR
We found that all of the T. thermophilus HB8 FadR binding sites were conserved in orthologous gene promoters in the highly related strain T. thermophilus HB27 (Table 3) with the exception of the TTHB017 promoter, which is present on a plasmid not present in the HB27 strain
Summary
Genome projects have yielded considerable information since the sequencing of the first whole microorganism genome, Haemophilus influenza, in 1995 [1,2]. Beyond a mere identification of open reading frames, it is important to determine the biological functions of encoded proteins and RNAs. One subset of proteins eliciting considerable interest is transcription factors, sequence-specific DNA-binding proteins that regulate transcription initiation, a major means of regulating gene expression. Genes encoding transcription factors are estimated to constitute, on average, ~5% of all protein-coding genes [3,4]. This reflects the need for prokaryotes to respond to a variety of changes in their environment necessitating a tight level of control over the expression of specific sets of genes, including.
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