Properties of P3 esters of nucleoside triphosphates as substrates for RNA polymerase from Escherichia coli.
P3-[(2,4-Dinitrophenyl)amino]ethyl (DNPNHEt) and P3-methyl phosphate esters of nucleoside 5'-triphosphates have been synthesized. Their properties as substrates in the initiation and elongation steps of transcription have been examined by using RNA polymerase from Escherichia coli and poly[d(A-T)] or T7 DNA as templates. It is shown that transcription can be initiated by ATP-EtNHDNP and that 2,4-dinitrophenyl residues are incorporated at the 5' end of the RNA molecules. Steady-state kinetic experiments of abortive initation on promoters A1 and A3 of T7 DNA revealed that ATP-EtNHDNP, ADP-EtNHDNP, and ATP-OCH3 have lower Km values and markedly reduced Vmax values compared to those of ATP. The two classes of esters, NTR-EtNHDNP and NTP-OCH3, were found to differ regarding their utilization as substrates for elongation. Both ATP-OCH3 and UTP-OCH3 are substrates for transcription. However, only the pyrimidine derivatives of NTP-EtNHDNP are elongation substrates which release DNPNHEt-PP upon utilization. This dramatic difference between the purine and pyrimidine derivatives of NTP-EtNHDNP reflects a selective process in the transcriptional complex for purines and pyrimidines.
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004136:Dinitrobenzenes / Q000138:chemical synthesis; D004926:Escherichia coli / Q000201:enzymology*; D007202:Indicators and Reagents; D007700:Kinetics; D009711:Nucleotides / Q000138:chemical synthesis*; D010755:Organophosphates / Q000138:chemical synthesis; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014158:Transcription, Genetic
D004136:Dinitrobenzenes; D007202:Indicators and Reagents; D009711:Nucleotides; D010755:Organophosphates; D012321:DNA-Directed RNA Polymerases