Ribozymes in Replication

The earliest forebears ofgenomes in a putative RNA World would likely have been simple ribozymes that could have replicated themselves from available materials. The earliest and simplest mechanism for replication would in turn likely have involved self-templated

Corresponding Author: Andrew D. Ellington—Department of Chemistry and Biochemistry, Center for Systems and Synthetic Biology University of Texas at Austin, Austin, Texas 78712, USA. Email: [email protected]

Figure 11.1. Nucleotide-based cofactors. Many cofactors are derived from adenosine nucleotides. R is the attachment point for various cofactor moieties to the ADP skeleton. In addition, many of the cofactors can be phosphorylated on the 3' hydroxyl (Y) or the 2' hydroxyl (X) groups of the ribose sugar.

Figure 11.1. Nucleotide-based cofactors. Many cofactors are derived from adenosine nucleotides. R is the attachment point for various cofactor moieties to the ADP skeleton. In addition, many of the cofactors can be phosphorylated on the 3' hydroxyl (Y) or the 2' hydroxyl (X) groups of the ribose sugar.

ligation via Watson-Crick base-pairing. In this regard, the templated replication ofshort oligonucleotides has actually been observed from trimeric or even shorter substrates.9,10 Assuming that such mechanisms could have yielded a nascent pool ofshort oligonucleotide precursors, extension and elaboration of this pool could have yielded longer oligomers with a range of improved catalytic activities. In particular, the formation ofmore complex RNA structures would have allowed improvements in ligation efficiency beyond simple templating.

As support for these hypotheses, we must go beyond the known natural ribozymes, which are few in number and have by and large evolved to catalyze RNA processing reactions rather than replication. Directed evolution offers an excellent means to recapitulate the RNA World by creating doppelgangers of early catalysts. The utility ofdirected evolution methods to inquire into origins was powerfully demonstrated by the evolution of a ribozyme that could catalyze the formation ofa 3'-5' phosphodiester bond at a ligation junction starting with a 5' triphosphorylated RNA as a substrate.11 This reaction is very similar to the one catalyzed by protein ligases and polymerases, although the ribozyme is much slower than its protein counterparts. This similarity was further exploited to select for a ribozyme that had polymerase activity using nucleoside triphosphates as substrates.12 Over four days of incubation this ribozyme could extend an RNA

primer that was annealed to it by six nucleotides, with a fidelity of ninety-two percent. However, the ability ofthis ribozyme to catalyze its own replication would have been grossly limited by the fact that to complete a second strand the ribozyme would eventually have had to unfold itself. To resolve this issue Bartel and coworkers attempted to evolve a polymerase which could recognize a detached (trans) template :primer complex.13,14 Through a complicated selection scheme involving a tethered substrate, they were in fact able to select for a variant of the original ribozyme that operated in trans and that demonstrated improved catalysis. This ribozyme was able to extend an RNA primer up to fourteen nucleotides in a twenty-four hour incubation with a fidelity over ninety-eight percent. Unfortunately, low polymerization efficiencies made longer syntheses impossible.

While the Bartel ligase and polymerase stand as avatars of a late RNA World that contained RNA polymerases, it seems unlikely that such complex structures would have arisen in an early RNA World. There must be molecular 'missing links' between the earliest, simplest self-replicating oligonucleotides and late stage polymerases (a 'march ofprogress' envisioned in Levy and Ellington, 2001;15 Fig. 11.2). One such missing link might be ribozyme polymerases that utilize oligo-nucleotides instead of mononucleotides as substrates. A number of small ribozyme ligases have been generated by directed evolution and the crystal structure ofone has recently been solved, giving us a glimpse of the chemistries involved in prebiotic replication.16 The 'march of progress' schema is further supported by computational simulations which showed that the evolution ofreplicators with greater efficiency and fidelity could be boot-strapped, leading to an increase in early genome size over time.17 An interesting caveat in these simulations was that the evolution of replicator complexity was directly related to the spatial separation ofthe replicators themselves. Populations of replicators that were allowed to diffuse freely in the computational grid did not evolve to higher complexity, while those that were spa

Figure 11.2. 'March of Progress' in ribozyme catalysis. This is one view of how primitive ribozyme replicators might have evolved increasing complexity over time. A) Simple replicators could have emerged from template-directed ligation of oligonucleotides; increasing elaboration would have led to (B) trans-acting ribozymes that catalyzed both the ligation of their own templates and other RNA molecules. C) To avoid parasitism, ribozymes might have taken advantage of short regions of sequence homology ('tags') to generate genomes. D) Long RNA stretches would have provided fodder for the evolution of template-directed polymerase ribozymes capable of rudimentary replication. Better polymerases could have emerged via a pathway similar to the directed evolution of the Bartel polymerase.10 This polymerase began as a ligase (i), which was then evolved into a nongeneral polymerase with an attached template (ii) and finally into a more general polymerase (iii). The advent of complex polymerases with increased fidelities and processivities would have allowed ribozymes and genomes with increasing sequence complexities to evolve, which could in turn have catalyzed the many biochemistries suspected to have been present in the RNA World (E). Ultimately, the invention of the translation apparatus would have led to the establishment of the modern DNA/protein World (F).

Figure 11.2. 'March of Progress' in ribozyme catalysis. This is one view of how primitive ribozyme replicators might have evolved increasing complexity over time. A) Simple replicators could have emerged from template-directed ligation of oligonucleotides; increasing elaboration would have led to (B) trans-acting ribozymes that catalyzed both the ligation of their own templates and other RNA molecules. C) To avoid parasitism, ribozymes might have taken advantage of short regions of sequence homology ('tags') to generate genomes. D) Long RNA stretches would have provided fodder for the evolution of template-directed polymerase ribozymes capable of rudimentary replication. Better polymerases could have emerged via a pathway similar to the directed evolution of the Bartel polymerase.10 This polymerase began as a ligase (i), which was then evolved into a nongeneral polymerase with an attached template (ii) and finally into a more general polymerase (iii). The advent of complex polymerases with increased fidelities and processivities would have allowed ribozymes and genomes with increasing sequence complexities to evolve, which could in turn have catalyzed the many biochemistries suspected to have been present in the RNA World (E). Ultimately, the invention of the translation apparatus would have led to the establishment of the modern DNA/protein World (F).

tially separated did. Thus, the evolution of replicators may have been intimately tied to the early cellularization of genomes.

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