title: Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.
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.
authors: Waksman Institute and Department of Chemistry and Chemical Biology, Rutgers University, 190 Frelinghuysen Road, Piscataway, NJ, 08854, USA.
authors: Anirban
authors: A
authors: Chakraborty
authors: Abhishek
authors: A
authors: Mazumder
authors: Miaoxin
authors: M
authors: Lin
authors: Adam
authors: A
authors: Hasemeyer
authors: Qumiao
authors: Q
authors: Xu
authors: Dongye
authors: D
authors: Wang
authors: Yon W
authors: YW
authors: Ebright
authors: Richard H
authors: RH
authors: Ebright
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001386:Azides; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D016296:Mutagenesis / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives; D010720:Phosphines; D010957:Plasmids / Q000235:genetics; D012808:Sigma Factor / Q000235:genetics
chemical_list: D000596:Amino Acids; D001386:Azides; D005456:Fluorescent Dyes; D010720:Phosphines; D012808:Sigma Factor; C027162:4-azidophenylalanine; D010649:Phenylalanine; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; C044646:phosphine