While it has become almost trivial to sequence whole genomes for all manner of organisms, fully understanding the biological roles of their encoded proteins remains far behind. This is especially true for transcription regulatory proteins, the primary gatekeeps of gene expression. Our laboratory has devised a biochemistry-centered, reverse-genetic approach to first determine consensus binding sites for these proteins, followed by bioinformatics analyses to identify genes potentially regulated by these proteins and their possible biological functions. Techniques involved included the novel combinatorial selection method Restriction Endonuclease Protection Selection and Amplification (REPSA), massively parallel semiconductor sequencing, biolayer interferometry-determined binding kinetics, and a host of bioinformatics packages including the MEME Suite, Softberry BPROM and Genome2D PePPER, analyses from ProOpDB and BioCyc, and databases at KEGG, NCBI, and UniProtKB. More importantly, all of the bench techniques are student-friendly, using safer reagents and feasible with limited time commitments. These modular projects expose students to a gamut of experiences, from microbiology and protein purification, through biochemistry/molecular biology analysis of protein-DNA complexes, to bioinformatics analyses of motifs, genomic sites, and biological functions. These should prove valuable in our student’s future endeavors. We have successfully used our REPSA-approach to characterize seven putative transcription factors from the model extreme thermophile Thermus thermophilus HB8, with undergraduate students being lead authors on resulting presentations and publications. Thus, this research may serve as a template for discovery-based undergraduate research experiences that can promote both scholarly and educational goals. 

Transcription factor discovery in the extremophile <i>Thermus thermophilus</i> HB8

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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Transcription factor discovery in the extremophile Thermus thermophilus HB8