Date of Award
Master of Science in Chemical Sciences (MSCB)
Dr. Michael Van Dyke
Dr. Thomas Leeper
Dr. Melanie Griffin
Transcription factor (TF) proteins act as molecular mechanisms that modulate the initiation of the first step in the expression of genes, gene transcription. Currently, knowledge of the DNA-binding specificities and genes regulated by many TFs, including those of well-studied model organisms such as Escherichia coli and Thermus thermophilus, remains incomplete or lacking which renders gaps in the understanding of the regulatory networks and systems biology of many organisms. Cyclic-AMP receptor protein (CRP) regulators and fumarate and nitrate reduction regulator (FNR) proteins compose the CRP/FNR superfamily of TFs, a diverse subgroup of TFs in bacteria which regulate various gene expression programs. In the present work, a reverse-genetic technique involving the combinatorial selection technique Restriction Endonuclease Protection, Selection, and Amplification (REPSA) has been applied to study TTHA1359, one of the four CRP/FNR superfamily TFs in the model organism T. thermophilus HB8. A TTHA1359-binding consensus, 5’-A(T/A)TGT(G/A)A(N6)T(C/T)ACA(A/T)T-3’, was identified using REPSA to select DNA sequences that TTHA1359 preferentially binds, massively parallel sequencing to acquire the sequence information of these selections, and bioinformatics to discover TTHA1359-binding motifs from the acquired sequence information. TTHA1359-binding to the identified consensus was biophysically characterized, and TTHA1359 was found to bind the identified consensus with high affinity, KD of ~ 3.4 nM. Several potential regulatory binding sites for TTHA1359 were identified bioinformatically by mapping the TTHA1359-binding consensus to the T. thermophilus HB8 genome. The findings of the present work should not only contribute to the knowledge of the DNA-binding specificity and genes regulated by TTHA1359 but also provide insight into the functionality of the applied reverse-genetic technique that should guide its future application to study other TFs.