Discovery of a DNA-binding Consensus and Potential Genomic Regulatory Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA1359

<p>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 <em>Escherichia coli</em> and <em>Thermus thermophilus,</em> 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 <em>T. thermophilus</em> 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, <em>K</em>_D of ~ 3.4 nM. Several potential regulatory binding sites for TTHA1359 were identified bioinformatically by mapping the TTHA1359-binding consensus to the <em>T. </em><em>thermophilus</em> 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.</p>

Name thesis:degree_name

Master of Science in Chemical Sciences (MSCB)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2020

Author dc:creator

• Teague, Josiah

Contributors dc:contributor

• Dr. Michael Van Dyke
• Dr. Thomas Leeper
• Dr. Melanie Griffin

• Transcription Factor
• Transcriptional Regulation
• Restriction Endonuclease Protection Selection and Amplification (REPSA)
• Thermus thermophilus HB8
• DNA-binding Specificity
• Chemistry

Repository record dc:identifier

https://digitalcommons.kennesaw.edu/mscs_etd/31

OAI identifier oai:identifier

oai:digitalcommons.kennesaw.edu:mscs_etd-1032