Direct coordination of nucleotide with the intrinsic metal in Escherichia coli RNA polymerase. A nuclear magnetic resonance study with cobalt-substituted enzyme.
Nuclear magnetic resonance studies were performed with Escherichia coli RNA polymerase (RPase) in which one of the two intrinsic Zn ions was substituted with Co(II) ion (Co-Zn RPase). The Co ion was located in the beta subunit which contains the initiation site of the enzyme. The paramagnetic effect of Co-Zn RPase on the relaxation rates of rapidly exchanging water protons indicated that the Co ion was accessible to solvent. There were approximately two water molecules in the inner coordination sphere of the Co ion, one of which could be replaced by the substrate adenosine 5'-triphosphate (ATP) or the initiator adenylyl-(3' leads to 5')-adenine (ApA) but to a much less extent by uridine 5'-triphosphate. The effects of ATP and ApA did not require the presence of DNA or Mg (II) ions, and their Kd values were estimated to be 0.15 and 0.075 mM, respectively. These results showed that the Co ion was at the initiation site. From the measurements of the paramagnetic effects of Co-Zn RPase on the relaxation rates of 1H and 31P nuclei of ATP, the distances from the intrinsic Co ion to H2, H8, and H1' were determined to be 4.1 +/- 0.6, 3.6 +/- 0.5, and 6.8 +/- 0.8 A, respectively, and those to the alpha-, beta-, and gamma-phosphorus atoms were 10.5 +/- 0.7, 15.1 +/- 1.1, and 14.1 +/- 0.8 A, respectively. These spatial relationships clearly indicate that the Co ion is directly coordinated to the base moiety of ATP bound at the initiation site. Thus, the intrinsic metal in the beta subunit of RNA polymerase may play a regulatory role in the recognition of the initiating nucleotide and may orient the nucleotide in a stereospecific position for the initiation.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate; D003035:Cobalt / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015226:Dinucleoside Phosphates; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics
D015226:Dinucleoside Phosphates; C026520:adenosine monophosphate-adenosine; D003035:Cobalt; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases