Photoaffinity modification of bacteriophage T7 DNA-dependent RNA polymerase by the reaction product containing the azido derivative of UTP

The possibility of using the 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)-propenyl-1]-UTP (N3-TFBP-UTP) as the affinity modificator of bacteriophage T7 DNA-dependent RNA polymerase was demonstrated. The UTP derivative used was rather efficient substrate substituting UTP in the transcription react...

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Bibliographic Details
Published inMolekuliarnaia biologiia Vol. 38; no. 6; p. 1059
Main Authors Tunitskaia, V L, Memelova, L V, Skoblov, Iu S, Kochetkov, S N
Format Journal Article
LanguageRussian
Published Russia (Federation) 01.11.2004
Subjects
Azides - chemistry
Bacteriophage T7 - enzymology
Chromatography, High Pressure Liquid
DNA-Directed RNA Polymerases - metabolism
Models, Molecular
Photoaffinity Labels
Uridine Triphosphate - chemistry
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Summary:The possibility of using the 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)-propenyl-1]-UTP (N3-TFBP-UTP) as the affinity modificator of bacteriophage T7 DNA-dependent RNA polymerase was demonstrated. The UTP derivative used was rather efficient substrate substituting UTP in the transcription reaction performed by the enzyme. The UV treatment of "stopped" reaction complex formed using three of four substrate ribonucleotides, allow to obtain the covalent binding between the enzyme and the reaction product of 9 nucleotides length. The isolation and the analysis of the obtained "nucleotide-peptide" showed that the sequence of modified peptide corresponded to the fragment Tyr802-Lys826, which belonged to the conservative motif C in the enzyme structure. His811 or Asp812 residues belonging to this sequence are most probable targets of the modification.
ISSN:0026-8984