Determination and Dissection of DNA-binding Specificity for the Thermus thermophilus HB8 Transcriptional Regulator TTHB099

<p>Transcription factors (TFs) have been extensively researched in certain well-studied organisms but far less so in others. Following the whole-genome sequencing of a new organism, TFs are typically identified through their homology with related proteins in other organisms. However, recent findings demonstrate that structurally similar TFs from distantly related bacteria are not usually evolutionary orthologs. Here we explore TTHB099, a cAMP receptor protein (CRP)-family TF from the extremophile <em>Thermus thermophilus</em> HB8. Using the <em>in vitro</em> iterative selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), we identified the preferred DNA-binding motif for TTHB099, 5'-TGT(A/g)n(t/c)c(t/c)(a/g)g(a/g)n(T/c)ACA-3', and mapped potential binding sites, and regulated genes within the <em>T. thermophilus</em> HB8 genome. Comparisons with expression profile data in TTHB099-deficient and wild type strains suggested that, unlike <em>E. coli</em> CRP (CRP_Ec), TTHB099 does not have a simple regulatory mechanism. However, we hypothesize that TTHB099 can be a dual-regulator similar to CRP_Ec.</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

• Moncja, Kristi

Contributors dc:contributor

• Dr. Michael Van Dyke
• Dr. Glen Meades
• Dr. Melanie Griffin

• transcription factors
• REPSA
• extremophile
• protein-DNA binding
• PCR
• type IIS restriction endonuclease
• biolayer interferometry (BLI)
• Biochemistry

Repository record dc:identifier

https://digitalcommons.kennesaw.edu/mscs_etd/35

OAI identifier oai:identifier

oai:digitalcommons.kennesaw.edu:mscs_etd-1036