Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.
Methods Mol Biol
; 1276: 101-31, 2015.
Article
en En
| MEDLINE
| ID: mdl-25665560
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN Polimerasas Dirigidas por ADN
/
Colorantes Fluorescentes
/
Aminoácidos
/
Biología Molecular
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2015
Tipo del documento:
Article
País de afiliación:
Estados Unidos