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Static and Kinetic Site-Specific Protein-DNA Photocrosslinking: Analysis of Bacterial Transcription Initiation Complexes

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DNA-Protein Interactions

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 543))

Summary

Static site-specific protein–DNA photocrosslinking permits identification of protein–DNA interactions within multiprotein–DNA complexes. Kinetic site-specific protein–DNA photocrosslinking – involving rapid-quench-flow mixing and pulsed-laser irradiation – permits elucidation of pathways and kinetics of formation of protein–DNA interactions within multiprotein–DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein–DNA photocrosslinking to bacterial transcription initiation complexes.

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Acknowledgments

The basic protocol for preparation of derivatized DNA fragments was developed by T. Lagrange, the basic protocol for preparation of RNAP was developed by H. Tang and K. Severinov, and the basic protocol for in-gel static photocrosslinking was developed by T.-K. Kim. We thank K. Severinov for plasmids; T.-K. Kim, T. Lagrange, D. Reinberg, and K. Severinov for discussion; and a Howard Hughes Medical Institute Investigatorship; and National Institutes of Health grant GM41376 to R.H.E. for financial support.

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  1. HHMI, Waksman Institute, Rutgers University, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA

    Nikolai Naryshkin, Sergei Druzhinin, Andrei Revyakin, Younggyu Kim, Vladimir Mekler & Richard H. Ebright

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  1. Nikolai Naryshkin
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  2. Sergei Druzhinin
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  3. Andrei Revyakin
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  4. Younggyu Kim
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  5. Vladimir Mekler
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  6. Richard H. Ebright
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Correspondence to Richard H. Ebright .

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    Naryshkin, N., Druzhinin, S., Revyakin, A., Kim, Y., Mekler, V., Ebright, R.H. (2009). Static and Kinetic Site-Specific Protein-DNA Photocrosslinking: Analysis of Bacterial Transcription Initiation Complexes. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_25

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    • DOI: https://doi.org/10.1007/978-1-60327-015-1_25

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