Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.
Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.
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DOI：
10.1007/978-1-4939-2392-2_6
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发表时间：
2015-01-01
期刊：
影响因子：
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通讯作者：
Ebright, Richard H
中科院分区：
文献类型：
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作者：
Chakraborty, Anirban;Mazumder, Abhishek;Ebright, Richard H
一种包括（1）用4-叠氮基-苯丙氨酸的非天然氨基酸诱变，（2）用探针-膦衍生物的Staudinger-Bertozzi连接，和（3）RNA聚合酶（RNAP）的体外重建的三步方法，使得能够有效地位点特异性掺入荧光探针、自旋标记、交联剂、切割剂、亲和标记或任何其它生物化学或生物物理探针，在RNAP的任何感兴趣的站点。该程序的直接扩展使得能够在RNAP的两个或更多个不同亚基中有效地位点特异性掺入两个或更多个不同的探针。我们目前的协议合成探针膦衍生物，制备RNAP亚基和转录起始因子σ，非天然氨基酸诱变RNAP亚基和σ，施陶丁格连接与非天然氨基酸含有RNAP亚基和σ，定量标记效率和标记特异性，和重建RNAP。
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 sigma, unnatural amino acid mutagenesis of RNAP subunits and sigma, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and sigma, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.