Substrate selection by RNA polymerase from E. coli. The role of ribose and 5'-triphosphate fragments, and nucleotides interaction.
Steady-state kinetic studies of the rifampicin-effected abortive initiation of transcription by E. coli RNA polymerase (EC 2.7.7.6) on the A1 T7 phage promoter were carried out with the use of ATP, UTP and a number of their appropriately modified analogues. The kinetic parameters KiA, KmB, Ki and KsB characterizing the affinity of the substrates and inhibitors of the reaction to the initiation and elongation sites of the enzyme:promoter and the enzyme:promoter:nucleoside triphosphate complexes were determined therefrom. Their comparative analysis indicated that 1) the triphosphate chain of the initiating purine nucleoside triphosphate interacts with some protein acceptor groups through the alpha- and beta-phosphate residues; the phosphates are engaged in binding of nucleoside triphosphates at the elongation site in the absence of the primer nucleotide; 2) the ribose 2'-OH of the elongating nucleotide, but neither of the ribose hydroxyl groups of the initiating nucleotide, participate in substrate recognition by protein receptors; 3) either substrate, ATP or UTP, bound to the initiation complex increases by about the same factor (greater than or equal to 10) the affinity of the other to its binding site; 4) the 3'-OH of the primer nucleotide and the gamma-phosphate of the elongating nucleotide are involved in the synergistic interaction of the substrates; alpha- and beta-phosphates of the elongating nucleotide, bound to some protein receptors, also contribute to this process. It is postulated that the interaction of substrates is mediated through an Mg2+ ion, known to be required for binding of the substrates in the elongation site, and a minimal molecular model of a PuoTP:Mg (II): nucleoside triphosphate chelate complex in the catalytic centre of the transcription initiation open complex is proposed.
D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism*; D012266:Ribose / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000187:drug effects; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; D000249:Adenosine Monophosphate; D012266:Ribose; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium; C026600:2'-deoxyadenosine triphosphate; D014544:Uridine Triphosphate