Arginine Cofactors on the Polymerase Ribozyme

The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor

Influence of arginine and amino modifications at the proximal end of the 5′-duplex, on polymerization.

<p>Shown is an autoradiogram of PAGE separated polymerization products. For each sample, the polymerization products at six incubation times are shown. The number of nucleotides added to the primer during polymerization is indicated. The eighth nucleotide addition results in two bands due to nucleotide misincorporation. The length of the 5′-duplex was 17 base pairs. No difference in polymerization efficiency was found between unmodified and modified ribozymes, within the errors of three replications of the experiments.</p

Influence of arginine and amino modifications on polymerization at low magnesium concentration.

<p>Quantitation of polymerization efficiencies for DNA conjugates at the ribozyme 5′-terminus (in the absence of a separate P2 oligo) and at the P2 site. The polymerization efficiency is described as the average number of nucleotides added per primer. For each experiment, three DNAs were tested: Unmodified (circles), amino modified (squares), and arginine modified (triangles) DNAs. Errors are standard deviations from three experiments.</p

Influence of arginine and amino modifications at the P2 oligo.

<p>The position and the modification of the DNA P2 oligo 5′-GGCGCC-3′ is shown for each reaction, as well as the number of nucleotides added to the primer. The images are representative for three experiments. (<b>A</b>) Autoradiogram of PAGE separated polymerization products, after 24 hours of polymerization. (<b>B</b>) Autoradiogram of PAGE separated polymerization products, after increasing times for polymerization.</p

Extension of primers that were base paired to the 5′-terminus of the polymerase ribozyme.

<p>The extension efficiency of these primers was measured as a function of the length of the 5′-duplex. (<b>A</b>) Autoradiogram of PAGE separated polymerization products, in the absence of the P2 oligo. For each length of the 5′-duplex (indicated) the unreacted primer and the reaction products are shown. (<b>B</b>) Quantitation of polymerization efficiencies. The polymerization efficiency was measured as the average number of nucleotides added per primer and plotted as a function of the length of the 5′-duplex. The polymerization efficiencies in the absence (open squares) and in the presence of the P2 oligo (filled squares) are shown. Errors are standard deviations from triplicate experiments and were usually smaller than the symbols.</p

Influence of arginine and amino modifications at the distal end of the 5′-duplex on polymerization.

<p>(<b>A</b>) Autoradiogram of PAGE separated polymerization products, with RNA/RNA duplexes at the 5′-terminus of the ribozyme, in the presence of our P2 oligo. The length of the 5′-duplexes as well as the chemical modification, are indicated. (<b>B</b>) Autoradiogram of PAGE separated polymerization products, with RNA/RNA duplexes at the 5′-terminus of the ribozyme, in the absence of a P2 oligo. The length of the 5′-duplexes as well as the chemical modification, are indicated. (<b>C</b>) Quantitation of polymerization efficiencies for RNA/RNA (filled symbols) and DNA/RNA (open symbols) duplexes at the ribozyme 5′-terminus. The polymerization efficiency is described as the average number of nucleotides added per primer. For each length of the 5′-duplex, three variants were tested: Unmodified (circles), amino modified (squares), and arginine modified (triangles) duplexes. Symbols above the grey dashed line show the results of reactions in the presence of the P2 oligo; symbols below the grey dashed line show the results in the absence of a P2 oligo. Errors are standard deviations from three experiments.</p

Structure of ribozyme constructs used in this study.

<p>The 5′-terminus of the ribozyme (green) is in close contact with the primer (red) and template (orange). The P2 oligo (dark blue) is base paired to a complementary region on the ribozyme (light blue), forming the P2 helix. (<b>A</b>) Secondary structure of the polymerase ribozyme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025030#pone.0025030-Johnston1" target="_blank">[7]</a> with the 5′-duplexes and the P2 duplex that were used to attach arginine or amino cofactors. The length of the 5′-duplex is indicated. “X” denotes the position of the chemical modification. The P2 oligo is truncated, and the internal mismatch was removed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025030#pone.0025030-Yao1" target="_blank">[12]</a>. (<b>B</b>) 3D structure of the ligase domain in the polymerase ribozyme, based on the crystal structure of the ligase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025030#pone.0025030-Shechner1" target="_blank">[27]</a>. Atoms that do not appear in the polymerase ribozyme were deleted. The asterisk denotes the position of the catalytic site. The positions where the 5′-duplex and the accessory domain are attached to the ligase domain are indicated, as well as the 5′-terminus and 3′-terminus of the P2 oligo.</p