Site-Specific Protein-DNA Photocrosslinking: Analysis of Bacterial Transcription Initiation Complexes
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1. |
Chemical (4 –6 ) and enzymatic (7 ) reactions are used to prepare a DNA fragment containing a photoactivatible crosslinking agent and an adjacent radiolabel incorporated at a single, defined DNA phosphate (with a 9.7 � linker between the photoreactive atom of the crosslinking agent and the phosphorus atom of the phosphate, and with an approximately 11 � maximum “reach” between potential crosslinking targets and the phosphorus atom of the phosphate).
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2. |
The multiprotein-DNA complex of interest is formed using the site-specifically derivatized DNA fragment, and the multiprotein-DNA complex is ultraviolet (UV)-irradiated, initiating covalent crosslinking with proteins in direct physical proximity to the photoactivatible crosslinking agent.
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3. |
Extensive nuclease digestion is performed, eliminating uncrosslinked DNA and converting crosslinked DNA to a crosslinked, radiolabeled 3–5 nucleotide “tag.”
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4. |
The “tagged” proteins are identified.
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