Inactivation of bacteriophage T7 DNA-dependent RNA polymerase by 5'-p-fluorosulfonylbenzoyladenosine. Identification of the modification site and the effect of the modification on enzyme action.
Bacteriophage T7 RNA polymerase was covalently modified by 5'-[4-fluorosulfonyl)benzoyl]adenosine (4-FSO2BzAdo). The modified enzyme lacks the ability to catalyze RNA synthesis from the phi 10 promoter of bacteriophage T7; both promoter and GTP binding being markedly decreased. The mild hydrolysis of the ester bond of 4-FSO2BzAdo within the covalent enzyme-inhibitor complex restores the RNA synthesis at a lower rate. Sequence studies show that Lys172 is the target of modification by 4-FSO2BzAdo. This residue, which is situated in the polypeptide region connecting two domains of RNA polymerase, was shown to be the primary site of the limited proteolysis occurring in vivo [Ikeda, R. A. & Richardson, C. C. (1987) J. Biol. Chem. 262, 3790-3799]. We propose that Lys172 is located outside the active site. Once this residue has reacted with 4-FSO2BzAdo, the nucleoside moiety of the analog is fixed in the NTP-binding site of the active centre and prevents binding of the substrates. Here, Lys172 per se is not important for the activity but serves as an 'anchor' for binding of the inhibitor.
V. A. Engelhardt Institute of Molecular Biology, USSR Academy of Sciences, Moscow.
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Tunitskaya
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AKh
Akbarov
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Luchin
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Memelova
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Rechinsky
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Kochetkov
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D008239:Lysine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D012313:RNA / Q000096:biosynthesis; D013604:T-Phages / Q000187:drug effects / Q000201:enzymology*; D014158:Transcription, Genetic; D014357:Trypsin
D010446:Peptide Fragments; D012313:RNA; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin; D008239:Lysine; D000241:Adenosine