Date of Award
Master of Science in Chemical Sciences (MSCB)
Dr. Michael Van Dyke
Dr. Carol Chrestensen
Dr. Tsai-Tien Tseng
In all organisms, genes exist that encode for regulatory proteins, called transcription factors (TFs) that can activate or repress transcription of specific genes depending on their biological function. Thermus thermophilus HB8 is speculated to contain 2,245 genes, of which 70 are postulated to be transcription factors. However, but for very few, little is known regarding the genes they regulate and their biological functions. The novel combinatorial method Restriction Endonuclease Protection, Selection, and Amplification (REPSA) has been successful for identifying and validating consensus binding sequences in T. thermophilus HB8 with previously studied TFs. Here REPSA was explored as a technique and method for identifying TTHA1437 and TTHA1719 TFs in T. thermophilus HB8. TTHA1437 REPSA results showed a promising DNA selection, but the results were not reproducible. Contrarily, TTHA1719 REPSA results did not show any selected DNAs, but during REPSA selections a rare DNA species, the asterisk species, was observed. REPA results for TTHA1437 showed nonspecific binding and TTHA1719 results showed no validation; EMSA also exhibited no DNA-ligand complex formation for both TFs. From the literature, a proposed potential consensus DNA for TTHA1719 proved promising with initial REPAs and a possible homologous DNA-binding consensus for TTHA1437 and E. coli CRP was explored but was unsuccessful. These results demonstrate that REPSA is not a viable method to characterize all TFs and the aim of this study was to determine why REPSA does not work to characterize these TFs in T. thermophilus HB8 and how it could be improved. Improvement on the REPSA method could consist of further DNA-binding assays concerning the proposed DNA-binding site(s) of TTHA1437 and TTHA1719 and other T. thermophilus HB8 TFs, specifically, before performing REPSA selections.