Site-Specific Protein-DNA Photocrosslinking: Analysis of Bacterial Transcription Initiation Complexes
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In work carried out in collaboration with the laboratory of D. Reinberg (University of Medicine and Dentistry of New Jersey), we have developed a site-specific protein-DNA photocrosslinking procedure to define positions of proteins relative to DNA in protein-DNA and multiprotein-DNA complexes (1 –3 ). The procedure has four parts Fig. 1 ):
| 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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Fig. 1. Site-specific protein-DNA photocrosslinking (1 –3 ). (A ,B ) Chemical and enzymatic reactions are used to prepare a full-length-promoter DNA fragment with a phenyl-azide photoactivatible crosslinking agent (R) and an adjacent radioactive phosphorus ~undefined) incorporated at a single, defined site. Based on the chemistry of incorporation, the maximum distance between the site of incorporation and the photoreactive atom is 9.7 �; the maximum distance between the site of incorporation and a crosslinked atom is approx 11 �. (C ) UV irradiation of the derivatized protein-DNA complex initiates crosslinking. Nuclease digestion eliminates uncrosslinked DNA and converts crosslinked, radiolabeled DNA to a crosslinked, radiolabeled 3–5 nucleotide “tag.”
