| Literature DB >> 25665560 |
Anirban Chakraborty1, Abhishek Mazumder, Miaoxin Lin, Adam Hasemeyer, Qumiao Xu, Dongye Wang, Yon W Ebright, Richard H Ebright.
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.Entities:
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Year: 2015 PMID： 25665560 PMCID： PMC4677679 DOI： 10.1007/978-1-4939-2392-2_6
Source DB: PubMed Journal: Methods Mol Biol ISSN： 1064-3745