Photoaffinity Modification of Bacteriophage T7 DNA-Dependent RNA Polymerase with a Reaction Product Containing an Azido Derivative of UTP

被引：0
作者：
V. L. Tunitskaya
L. V. Memelova
Yu. S. Skoblov
S. N. Kochetkov
机构：
[1] Russian Academy of Sciences,Engelhardt Institute of Molecular Biology
来源：
Molecular Biology | 2004年 / 38卷
关键词：
bacteriophage T7; DNA-dependent RNA polymerase; photoaffinity modification; azido derivatives of nucleotides;
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摘要：
The possibility of using 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)-propenyl-1]-UTP (N3-TFBP-UTP) as a photoaffinity modifier of T7 DNA-dependent RNA polymerase (RNAP) was demonstrated. The UTP derivative proved to act as an effective substrate substituting UTP in transcription driven by T7 RNAP. A reaction complex obtained in the presence of all but one ribonucleotide substrate was UV-irradiated, and covalent binding between the enzyme and the reaction product (a 9-mer oligoribonucleotide) was detected. Isolation and analysis of the relevant nucleotide–peptide showed that affinity modification involved the peptide Tyr802–Lys826, which belongs to the conserved motif C of T7 RNAP. Functionally important His811 and Arg812 were assumed to be the targets of modification.
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页码：907 / 913
页数：6
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