Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.

Chakraborty, A, Mazumder A, Lin M, Hasemeyer A, Xu Q, Wang D, Ebright YW, Ebright RH.  2015.  

Journal:

Methods in molecular biology (Clifton, N.J.)

Volume Number:

1276

Pages:

101-31

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.

Citation:
Chakraborty, A, Mazumder A, Lin M, Hasemeyer A, Xu Q, Wang D, Ebright YW, Ebright RH.  2015.  Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.. Methods in molecular biology (Clifton, N.J.). 1276:101-31.