MAPPING OF THE ACTIVE-SITE OF T7 RNA-POLYMERASE WITH 8-AZIDO ATP

被引:15
作者
KNOLL, DA [1 ]
WOODY, RW [1 ]
WOODY, AYM [1 ]
机构
[1] COLORADO STATE UNIV,DEPT BIOCHEM,FT COLLINS,CO 80523
关键词
T7 RNA POLYMERASE; PHOTOAFFINITY LABELING; 8-AZIDO ATP; ACTIVE-SITE MAPPING;
D O I
10.1016/0167-4838(92)90154-6
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The photoaffinity analog of ATP, 8-azidoATP, labels T7 RNA polymerase. Photoincorporation exhibits saturation behavior and is protected against by the substrate ATP. 8-AzidoATP is a competitive inhibitor of ATP incorporation with K(i) approximately 40-mu-M. The photolabeled T7 RNA polymerase, following cyanogen bromide digestion, was analyzed by phenylboronate agarose column chromatography followed by reverse-phase high pressure liquid chromatography. Sequencing of the peptides labeled with radioactive photoprobe allowed the identification of three peptides, P314-M362 (I), L550-M666 (II), and F751-M861 (III). These peptides are in the proximity of the photoprobe 8-azidoATP and, therefore, expected to contain functionally significant residues and define an active site domain. These peptides (I and II) contain residues previously implicated in T7 RNA polymerase activity or show homology to active site regions of the Klenow fragment of DNA polymerase I (II and III).
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页码:252 / 260
页数:9
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