Abstract
A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger–Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.
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Acknowledgements
We thank Peter Schultz and Ryan Mehl for plasmids. This work was supported by National Institutes of Health grant GM041376 and a Howard Hughes Medical Institute Investigatorship to R.H.E.
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Chakraborty, A. et al. (2015). Site-Specific Incorporation of Probes into RNA Polymerase by Unnatural-Amino-Acid Mutagenesis and Staudinger–Bertozzi Ligation. In: Artsimovitch, I., Santangelo, T. (eds) Bacterial Transcriptional Control. Methods in Molecular Biology, vol 1276. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2392-2_6
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DOI: https://doi.org/10.1007/978-1-4939-2392-2_6
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