Synthesis and properties of adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid)naphthyl ethylamidate: a fluorescent nucleotide substrate for DNA-dependent RNA polymerase from Escherichia coli.
A new fluorescent ATP analog, adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid)naphthyl ethylamidate (gamma-1,5-EDANS)ATP, containing the fluorophore N-(aminoethyl)-5-naphthylamine-1-sulfonic acid attached via a gamma-phosphoamidate bond was synthesized in good yield. It has absorption maxima at 255 and 344 nm and a fluorescence emission maximum at 490 nm. These spectral characteristics permit its uses as an energy acceptor for energy transfer from the intrinsic protein fluorophores and as an energy donor for the energy transfer to the intrinsic Co of Co-substituted RNA polymerases. This analog is a good substrate for Escherichia coli RNA polymerase and can be used to initiate the RNA chain. Incorporation of this analog into the total RNA synthesized was about 60% of that observed for ATP, independent of the templates used. Its Km values (22 and 118 microM) are twofold higher and its Vmax values (45 and 59 nmol/min/mg of enzyme) are 40% lower than those for ATP using calf thymus DNA and poly[d(A-T)], respectively, as template. For abortive initiation reaction using pAR1435 plasmid DNA as template, the Km and Vmax values of this analog are 2.7 times higher and 7 times lower, respectively, than those of ATP. With its desirable spectroscopic properties, (gamma-1,5-EDANS)ATP is a good probe for the studies of nucleotide-protein interactions, active site mapping of RNA polymerase, and other ATP-utilizing biological systems.
F Y
FY
Wu
A W
AW
Abdulwajid
D
D
Solaiman
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D007700:Kinetics; D012329:RNA, Bacterial / Q000096:biosynthesis; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D012329:RNA, Bacterial; C048716:adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid)naphthyl ethylamidate; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases