Since a major function of RNA polymerase must be to bring together substrates in the optimal configuration for internucleotide bond formation, studies have been undertaken to understand the geometrical relationship of the two substrates. A model has been constructed for the geometry of interaction of two ATP molecules poised on the active site of the Escherichia coli enzyme for the formation of the first bond in RNA synthesis. The model is based primarily on the distance, measured by EPR, between the two metals in the i and i + 1 subsites, as well as distances, measured by NMR, from each metal to points on the substrate in the same subsite, in the presence of a poly(dAdT).poly(dAdT) template. Both the Zn(II) in the i site and the Mg(II) in i + 1 are displaced by Mn(II). The nucleotide bases are not parallel to each other, in line with the reaction of the ATP molecules with DNA within the transcription bubble. The metal in the i site appears too far removed from substrate to participate in catalysis, but the metal in i + 1 is in position to bind to the beta- and gamma-phosphate groups and probably is involved in cleavage of the triphosphate, as has been previously suggested.

The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.

Distances between the metal ions bound to the product terminus i site and the substrate i+l site ofEscherichia col RNA polymerase range from 5.0 to 5.6 A when the substrate is complementary to a template base and from 6.5 to 7.0 A for a noncomplementary relationship. The metal bound to the substrate at the i+1 site exhibits a constant distance to the three phosphates on the substrate regardless of complementarity, but the dice to base and ribose protons changes.The differences in these geometric parameters are explained by the ability of the enzyme to assume two conformations, one to place correct nucleotide substrates in optimal position for bond formation and the other to prevent incorrect nucleotides from assuming such a position. In this scheme a metal-riphosphate complex can move toward or away from the terminal 3' OH group of the growing RNA chain, to assure fidelity of transcription.The primary process responsible for ensuring the fidelity of transcription is the Watson-Crick base pairing between the DNA base to be copied and the base on the incoming nucleoside triphosphate (NTP). This process depends on the greater stability of Watson-Crick base pairs compared to others; yet the difference in stability is insufficient to account for the observed fidelity oftranscription (1-5). Consequently, there must be another factor in producing this fidelity. The enzyme RNA polymerase is present and required at the point of elongation, where an incoming NTP enters the system to be incorporated into the growing RNA chain. This enzyme is a versatile one that has a variety of known functions. To enhance fidelity, the enzyme would have to engage in another hitherto unknown function: to distinguish between NTPs with "correct" and "incorrect" bases and reject the incorrect bases, thus enhancing the incorporation of the correct bases. We have evidence that the enzyme performs this function through its ability to assume two conformations, one to promote elongation with the incoming NTP and the other to prevent it. To determine whether the RNA polymerase is performing in this way, we have been studying the relationship between two NTPs in the active site of the Escherichia coli enzyme (6, 7), one in the i site (where the growing RNA chain is located) and the other in the i+ 1 site (for the incoming NTP) (8). We are thus looking at a system poised for the formation of the first bond in RNA synthesis, leading to the dinucleotide in which the first two bases of the DNA have been transcribed. In our previous studies (6, 7), we have determined the geometric relationship between ATP substrates in both i and i+1 sites with poly(dAdT)'poly(dAdT) serving as a template (6, 7). We accomplished this by measuring distances by electron paramagnetic resonance (EPR) between the Zn(II) and Mg(II) ions in the two sites, both replaced by Mn(II). We then used distances measured in our laboratory (7) and elsewhere (9-11) between these metals and points on the two substrates to produce a model for the geometry between the two ATP molecules...

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