Study of Transcription Factor- DNA interaction using AFM

POSTER

Abstract

This research aims to determine the characteristics of transcription factor binding to DNA using Atomic Force Microscopy. Atomic Force Microscopy (AFM) is an instrument that scans extremely fine surface features to create three-dimensional images. AFM can also be used to measure the binding force between a protein anchored on a solid support and a small molecule attached to the AFM tip In this study, we are examining, CRP, a transcription factor found in \textit{E coli,} and its association with known CRP promoter regions in the DNA. CRP protein with T8C mutation was isolated using His tag purification method. The cysteine mutation in the 8$^{\mathrm{th}}$ position of the primary structure of the protein will be used to attach CRP to an ultra-flat gold surface through a thiol bond. This technique results in the formation of a CRP monolayer, which allows us to image the protein and perform force measurements. Segments of DNA that contain the promoter region for CRP were amplified by PCR reaction from chromosomal DNA of E. coli bacteria strain W3110, imaged and analyzed by ezAFM. Future work will include attaching this promoter region to the AFM tip and measuring the binding for between the CRP on the solid support and the DNA on the tip.

Authors

  • Khdr Eskandar

    Monmouth College, IL

  • Swapan Chattopadhyay

    Gravitational Astrophysics Lab, NASA Goddard Space Flight Center, Northern Illinois University and Argonne National Laboratory, Northern Illinois University, None, Monmouth College, IL, University of California Santa Cruz, Southern Illinois University Carbondale, Argonne National Laboratory, University of Iowa, Sinop University, Bingol University, Univ of Northern Iowa, University of California, Riverside, University of Northern Iowa, University of Wisconsin, Fairfield University, North Central College

  • Swapan Chattopadhyay

    Gravitational Astrophysics Lab, NASA Goddard Space Flight Center, Northern Illinois University and Argonne National Laboratory, Northern Illinois University, None, Monmouth College, IL, University of California Santa Cruz, Southern Illinois University Carbondale, Argonne National Laboratory, University of Iowa, Sinop University, Bingol University, Univ of Northern Iowa, University of California, Riverside, University of Northern Iowa, University of Wisconsin, Fairfield University, North Central College

  • Swapan Chattopadhyay

    Gravitational Astrophysics Lab, NASA Goddard Space Flight Center, Northern Illinois University and Argonne National Laboratory, Northern Illinois University, None, Monmouth College, IL, University of California Santa Cruz, Southern Illinois University Carbondale, Argonne National Laboratory, University of Iowa, Sinop University, Bingol University, Univ of Northern Iowa, University of California, Riverside, University of Northern Iowa, University of Wisconsin, Fairfield University, North Central College