Direct coordination of nucleotide with the intrinsic metal in Escherichia coli RNA polymerase. A nuclear magnetic resonance study with cobalt-substituted enzyme

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Abstract
NMR studies were performed with E. coli RNA polymerase (RPase) in which 1 of the 2 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 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 2 water molecules in the inner coordination sphere of the Co ion, one of which could be replaced by the substrate ATP or the initiator adenylyl-(3'' .fwdarw. 5'')-adenine (ApA) but to a much less extent by UTP. The effects of ATP and ApA did not require the presence of DNA or Mg(II) ions, and their Kd values were 0.15 and 0.075 mM, respectively. The Co ion was apparently 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 .ANG., respectively, and those to the .alpha.- and .beta.- and .gamma.-phosphorus atoms were 10.5 .+-. 0.7, 15.1 .+-. 1.1 and 14.1 .+-. 0.8 .ANG., respectively. These spatial relationships clearly indicate that the Co ions 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.