Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and staudinger-bertozzi ligation

Anirban Chakraborty; Abhishek Mazumder; Miaoxin Lin; Adam Hasemeyer; Qumiao Xu; Dongye Wang; Yon W. Ebright; Richard H. Ebright

doi:10.1007/978-1-4939-2392-2_6

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and staudinger-bertozzi ligation

Anirban Chakraborty, Abhishek Mazumder, Miaoxin Lin, Adam Hasemeyer, Qumiao Xu, Dongye Wang, Yon W. Ebright, Richard H. Ebright

Rutgers, The State University

Research output: Chapter in Book/Report/Conference proceeding › Chapter

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.

Original language	American English
Title of host publication	Bacterial Transcriptional Control
Subtitle of host publication	Methods and Protocols
Publisher	Springer New York
Pages	101-131
Number of pages	31
ISBN (Electronic)	9781493923922
ISBN (Print)	9781493923915
DOIs	https://doi.org/10.1007/978-1-4939-2392-2_6
State	Published - Feb 9 2015

ASJC Scopus subject areas

General Immunology and Microbiology
General Biochemistry, Genetics and Molecular Biology
General Medicine

Keywords

4-Azido-phenylalanine
Alexa 647
Alexa 647-phosphine
Cy3B
Cy3B-phosphine
Fluorescent probes
In vitro reconstitution
Phosphines
RNA polymerase
RNA polymerase α subunit
RNA polymerase β subunit
RNA polymerase β' subunit
RNA polymerase ω subunit
Staudinger ligation
Unnatural-amino-acid mutagenesis
σ70

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10.1007/978-1-4939-2392-2_6

Cite this

Chakraborty, A., Mazumder, A., Lin, M., Hasemeyer, A., Xu, Q., Wang, D., Ebright, Y. W., & Ebright, R. H. (2015). Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and staudinger-bertozzi ligation. In Bacterial Transcriptional Control: Methods and Protocols (pp. 101-131). Springer New York. https://doi.org/10.1007/978-1-4939-2392-2_6

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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.",

keywords = "4-Azido-phenylalanine, Alexa 647, Alexa 647-phosphine, Cy3B, Cy3B-phosphine, Fluorescent probes, In vitro reconstitution, Phosphines, RNA polymerase, RNA polymerase α subunit, RNA polymerase β subunit, RNA polymerase β' subunit, RNA polymerase ω subunit, Staudinger ligation, Unnatural-amino-acid mutagenesis, σ70",

author = "Anirban Chakraborty and Abhishek Mazumder and Miaoxin Lin and Adam Hasemeyer and Qumiao Xu and Dongye Wang and Ebright, {Yon W.} and Ebright, {Richard H.}",

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doi = "10.1007/978-1-4939-2392-2_6",

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AU - Chakraborty, Anirban

AU - Mazumder, Abhishek

AU - Lin, Miaoxin

AU - Hasemeyer, Adam

AU - Xu, Qumiao

AU - Wang, Dongye

AU - Ebright, Yon W.

AU - Ebright, Richard H.

N1 - Publisher Copyright: © Springer Science+Business Media New York 2015. All rights are reserved.

PY - 2015/2/9

Y1 - 2015/2/9

N2 - 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.

AB - 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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KW - Alexa 647

KW - Alexa 647-phosphine

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KW - Phosphines

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KW - RNA polymerase α subunit

KW - RNA polymerase β subunit

KW - RNA polymerase β' subunit

KW - RNA polymerase ω subunit

KW - Staudinger ligation

KW - Unnatural-amino-acid mutagenesis

KW - σ70

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SN - 9781493923915

SP - 101

EP - 131

BT - Bacterial Transcriptional Control

PB - Springer New York

ER -

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and staudinger-bertozzi ligation

Abstract

ASJC Scopus subject areas

Keywords

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