Academic department under which the project should be listed

Chemistry and Biochemistry

Faculty Sponsor Name

Michael Van Dyke

Project Type

Oral Presentation (15-min time slots)

Abstract (250 words maximum)

The function of many genes and the biological roles of their encoded products are still not well characterized. Given the sequence-specific DNA-binding properties of transcription factor proteins, it is possible to purify them, identify the responsible polypeptide(s), determine their consensus binding sequences, and identify their genomic binding sites. Thus, one can go from cellular extract to proposed biological regulatory roles in relatively short order. Our goal is to identify and characterize orphan DNA-binding proteins in the model organism E. coli K12 using the novel combinatorial technique, REPSA (Restriction Endonuclease Protection Selection Amplification), as well as further develop the REPSA to be able to analyze whole cell extracts. REPSA does not require any prior knowledge of a ligand in order to determine its preferred binding site on duplex DNA and has been previously utilized successfully to identify binding specificity. We are currently studying LexA, a repressor protein with a defined consensus binding sequence, although a technique such as REPSA has never been used to study its specificity. Thus, LexA provides an appropriate starting point to optimize REPSA and compare its data with previously accepted findings. Even if novel sequences are not found, our studies will provide a proof-of-concept and methodology applicable to more uncharacterized transcription factors in E. coli and other organisms. This research is expected to lead to a greater understanding of bacterial biology at a molecular level and ultimately advance public health by characterizing orphan regulatory proteins that can be critical players in many different microbial diseases and the inner workings of the human microbiome.

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REPSA-Directed Identification of DNA-Binding Specificity for Orphan Transcription Factors

The function of many genes and the biological roles of their encoded products are still not well characterized. Given the sequence-specific DNA-binding properties of transcription factor proteins, it is possible to purify them, identify the responsible polypeptide(s), determine their consensus binding sequences, and identify their genomic binding sites. Thus, one can go from cellular extract to proposed biological regulatory roles in relatively short order. Our goal is to identify and characterize orphan DNA-binding proteins in the model organism E. coli K12 using the novel combinatorial technique, REPSA (Restriction Endonuclease Protection Selection Amplification), as well as further develop the REPSA to be able to analyze whole cell extracts. REPSA does not require any prior knowledge of a ligand in order to determine its preferred binding site on duplex DNA and has been previously utilized successfully to identify binding specificity. We are currently studying LexA, a repressor protein with a defined consensus binding sequence, although a technique such as REPSA has never been used to study its specificity. Thus, LexA provides an appropriate starting point to optimize REPSA and compare its data with previously accepted findings. Even if novel sequences are not found, our studies will provide a proof-of-concept and methodology applicable to more uncharacterized transcription factors in E. coli and other organisms. This research is expected to lead to a greater understanding of bacterial biology at a molecular level and ultimately advance public health by characterizing orphan regulatory proteins that can be critical players in many different microbial diseases and the inner workings of the human microbiome.