A Step towards Modeling the RNA World
Ribozyme coped with the synthesis of its ancestor
Vera Mukhina, N+1
With the help of directed evolution, scientists have derived a ribozyme that is able to synthesize its ancestor, a shorter RNA molecule that also has catalytic activity. This work, published in the journal Proceedings of the National Academy of Sciences (Tjhung et al., An RNA polymerase ribozyme that synthesizes its own ancestor), brings closer the reconstruction of self-replicating RNA systems, which – if we believe the hypothesis of the RNA world - gave rise to all life on Earth.
Obtaining such systems in vitro could be considered a significant confirmation of the hypothesis of the RNA world postulating the emergence of life from self-reproducing RNA forms. A lot has been done in this direction in recent years, but it is not yet possible to grow a fully self-reproducing system with the help of directed evolution. To do this, the ribozyme synthesizing RNA – RNA polymerase - must be able to accurately synthesize both its own sequence and a complementary one to use it as a matrix. Some in vitro grown ribozymes already know how to copy themselves partially or even completely, but these reactions depend on the selection of substrates-oligonucleotides that limit the ability of synthesized RNA to mutations and evolution.
Katrina Jung and her colleagues from the Salk Institute managed to grow an RNA polymerase that is able to synthesize from mononucleotides, though not itself, but the ancestral ribozyme. The already known polymerase 24-3 was taken as a basis. It was obtained earlier from other ribozymes and it was already able to copy small RNA sequences. The researchers set out to evolve this enzyme so that it could synthesize complex ribozymes, and as an intermediate goal they decided to achieve the synthesis of ribozyme-an endonuclease capable of cutting RNA.
To "train" the polymerase, they used the method of directed evolution. Slightly mutated sequences of the original polymerase were attached to the surface with a primer, and then allowed to work. If she coped with the work and synthesized a normal endonuclease, she cut it off from the surface, and if not, the polymerase remained attached. The researchers, who got into the polymerase solution, multiplied with the addition of new mutations and then repeated the cycle with them, but giving less time to work. Thus, only functional polymerases were selected to a new level, and with each cycle they coped with the task more quickly. In total, the scientists conducted 38 cycles, but more or less effective polymerases appeared already on the 14th round. In the course of evolution, the researchers obtained three families of ribozymes, of which polymerase 38-6 proved to be the most effective. It was distinguished from the original sequence of polymerase 24-3 by 14 mutations.
The scheme of operation of the finished polymerase. At the first stage, a primer attached to the surface is sewn to it. Then its sequence is completed according to the RNA sample. The freshly synthesized sequence collapses into a hammer-like structure with its own enzymatic sequence and bites off the polymerase. A drawing from an article in PNAS.
Having made sure that the polymerase was working properly, the authors of the article set her the task of synthesizing another ribozyme, a class I ligase. It was not chosen by chance. In fact, this ligase is a simple ancestor of the polymerase itself. They have a common catalytic core, 77 percent of whose sequences they have the same.
Despite the success with endonuclease, in this case, ribozyme 38-6 had problems with accuracy. The ligase consists of about one hundred ribonucleotides, and accuracy of at least 99 percent is required for its error-free synthesis. But polymerase 38-6 only managed to substitute the correct ribonucleotide in 83 percent of cases. This is clearly not enough for the full-fledged synthesis of the ligase, and even more so for the synthesis of the polymerase itself, whose length is twice as long.
To simplify the task, the researchers disassembled the ligase into three separate sequences capable of assembling into a single working structure. Such a complex is slightly inferior in efficiency to a conventional ligase, but it is much easier to synthesize it without errors.
RNA ligase, each of the three chains of which was synthesized by ribozyme polymerase.
Parallel synthesis of three chains has been successful, and, according to scientists, it is the longest and most complex ribozyme synthesized from individual mononucleotides. The authors of the article suggest that further improvements in the system can tighten the accuracy of the ribozyme to a level at which its normal self-replication will be possible.
The tasks of this study included the creation of a self–replicating ribozyme, while the assembly of the bricks for its posturing – nucleosides - remained beyond its limits. Meanwhile, they are absolutely necessary for synthesis and it is still not fully clear where prehistoric ribozymes took them from. Several methods of synthesis of each nucleoside have already been proposed, but they turned out to be incompatible with each other. In a recent study, scientists suggested that the answer lies in the cyclic change of humidity, due to which suitable conditions for the synthesis of different nucleosides alternated.
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