Abstract
A better understanding of the structural basis for the preferences of RNA and DNA polymerases for nucleoside-5'-triphosphates (NTPs) could help define the catalytic mechanisms for nucleotidyl transfer during RNA and DNA synthesis and the origin of primordial nucleic acid biosynthesis. We show here that ribonucleoside-5'-diphosphates (NDPs) can be utilized as substrates by RNA polymerase (RNAP). We found that NDP incorporation is template-specific and that noncognate NDPs are not incorporated. Compared with the natural RNAP substrates, NTPs, the Km of RNAP for NDPs was increased ∼4-fold, whereas the V max was decreased ∼200-fold. These properties could be accounted for by molecular modeling of NTP/RNAP co-crystal structures. This finding suggested that the terminal phosphate residue in NTP (not present in NDP) is important for positioning the nucleotide for nucleolytic attack in the nucleotidyl transfer reaction. Strikingly, a mutational substitution of the active-center βR1106 side chain involved in NTP positioning also strongly inhibited NDP-directed synthesis, even though this residue does not contact NDP. Substitutions in the structurally analogous side chain in RB69 DNA polymerase (Arg-482) and HIV reverse transcriptase (Lys-65) were previously observed to inhibit dNDP incorporation. The unexpected involvement of these residues suggests that they affect a step in catalysis common for nucleic acid polymerases. The substrate activity of NDPs with RNAP along with those reported for DNA polymerases reinforces the hypothesis that NDPs may have been used for nucleic acid biosynthesis by primordial enzymes, whose evolution then led to the use of the more complex triphosphate derivatives.
Highlights
A better understanding of the structural basis for the preferences of RNA and DNA polymerases for nucleoside-5-triphosphates (NTPs) could help define the catalytic mechanisms for nucleotidyl transfer during RNA and DNA synthesis and the origin of primordial nucleic acid biosynthesis
The substrate activity of NDPs with RNA polymerase (RNAP) along with those reported for DNA polymerases reinforces the hypothesis that NDPs may have been used for nucleic acid biosynthesis by primordial enzymes, whose evolution led to the use of the more complex triphosphate derivatives
We first asked if NDPs could extend RNA primers in ternary elongation complexes (TEC)2 assembled from RNAP and synthetic oligonucleotides
Summary
A better understanding of the structural basis for the preferences of RNA and DNA polymerases for nucleoside-5-triphosphates (NTPs) could help define the catalytic mechanisms for nucleotidyl transfer during RNA and DNA synthesis and the origin of primordial nucleic acid biosynthesis. The substrate activity of NDPs with RNAP along with those reported for DNA polymerases reinforces the hypothesis that NDPs may have been used for nucleic acid biosynthesis by primordial enzymes, whose evolution led to the use of the more complex triphosphate derivatives. Both NTPs and NDPs possess phosphoanhydride bonds that provide energy to direct biochemical reactions through coupling mechanisms. This is supported by the ability of a currently existing enzyme, polynucleotide phosphorylase (PNP) to utilize efficiently NDP for non–template-encoded RNA synthesis
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