用于分析大肠杆菌RNA聚合酶底物结合位点的UTP光亲和类似物5-叠氮基-UTP的特性研究

Characterization of a photoaffinity analog of UTP, 5-azido-UTP for analysis of the substrate binding site on E. coli RNA polymerase.

作者信息

Woody A Y, Evans R K, Woody R W

机构信息

Department of Biochemistry, Colorado State University, Fort Collins 80523.

出版信息

Biochem Biophys Res Commun. 1988 Feb 15;150(3):917-24. doi: 10.1016/0006-291x(88)90716-4.

DOI:10.1016/0006-291x(88)90716-4

PMID:2449209

Abstract

The substrate binding site on E. coli RNA polymerase was investigated by photoaffinity labeling with a photoaffinity analog of UTP, 5-azido-UTP. We have established that 5-azido-UTP is a substrate for RNA polymerase by specific transcription on 229 bp DNA containing the gene II promoter of M13 phage. Analysis of the initial rate of RNA synthesis gives Km(5-azido-UTP) approximately 80 microM. Photolabeling with varying concentrations of 5-azido-UTP follows a saturation curve with the midpoint occurring at a 5-azido-UTP concentration of 65 microM near to the Km obtained by kinetic analysis. 5-Azido-UTP photolabels the beta', beta, and sigma subunits to about the same extent, both in the presence (33, 31, and 36%) and absence (35, 30 and 35%) of DNA. This labeling pattern is somewhat different from that obtained with 8-azido-ATP (beta' greater than sigma much greater than beta greater than alpha).

摘要

利用尿苷三磷酸（UTP）的光亲和类似物5-叠氮基-UTP进行光亲和标记，对大肠杆菌RNA聚合酶上的底物结合位点进行了研究。我们通过在含有M13噬菌体基因II启动子的229 bp DNA上进行特异性转录，证实5-叠氮基-UTP是RNA聚合酶的一种底物。对RNA合成初始速率的分析得出，Km（5-叠氮基-UTP）约为80微摩尔。用不同浓度的5-叠氮基-UTP进行光标记，得到一条饱和曲线，中点出现在5-叠氮基-UTP浓度为65微摩尔处，接近通过动力学分析获得的Km值。在有DNA（33%、31%和36%）和无DNA（35%、30%和35%）的情况下，5-叠氮基-UTP对β'、β和σ亚基的光标记程度大致相同。这种标记模式与用8-叠氮基-ATP获得的标记模式（β'＞σ＞β＞α）有所不同。