Ribozymes Amino Acids and the Advent ofTranslation

The advent of an efficient translational apparatus would have been the pinnacle of biochemical evolution in the RNA World and also its downfall. Efficient translation would have led to longer peptides which could have acted as powerful catalysts. Since protein catalysts have many advantages over ribozymes, once they began to be translated from heritable, genomic RNAs they would have also begun to evolve and to replace their less efficient RNA counterparts. Nonetheless, at the outset highly evolved ribozymes would still have been more efficient than nascent and unevolved proteins. It is therefore likely that the some of the earliest products of translation would have been short peptides that would have acted as cofactors to supply chemical functionalities otherwise unavailable to RNAs (and in turn generating the earliest ribonucleoproteins).

These insights may speak to one of the enduring mysteries of molecular biology: why did the translation apparatus evolve in the first place ? From one vantage, translation looks like a textbook example of that well-known oxymoron, anticipatory evolution (i.e., ribozymes were the primary catalysts in a hypothetical early cell and yet somehow knew to create an apparatus to replace themselves). Even if the first translated peptides augmented ribozyme-mediated catalysis, there must have been some simple and obvious evolutionary advantage to augmenting catalysis prior to inventing a complex apparatus to do so. One possible explanation is that since amino acids would have been available in the prebiotic soup, ribozymes could have originally used these simple and available compounds as cofactors. Further increases in catalytic precision and complexity would have driven the conjugation and polymerization of amino acids in series and chains. As an example of how translated peptides could have functioned with RNA catalysts, peptide dependant ribozymes have been selected that activate ligation by >18,000 fold.27 These experiments also demonstrate that in addition to acting as potential cofactors, peptides could potentially have acted as allosteric effectors regulating metabolism in the RNA World.

These musings in turn beg the question of how amino acids would have been utilized by RNA catalysts. Our discussion ofcofac-tors above provides a possible answer: amino acids could have been either noncovalently bound or covalently appended to ribozymes themselves. Both possibilities can be supported experimentally. Roth and Breaker selected a deoxyribozyme that utilized a noncovalently bound histidine as a cofactor to catalyze the cleavage of an RNA substrate.28 The choice ofhistidine in these experiments was particularly interesting as histidine is the only amino acid that can function in general acid or base catalysis at physiological pH values (—7.0). The use ofthe imidazole sidechain of hisidine for this function is of course widespread in proteins and would have been especially useful for nucleic acid catalysts as they lack facile proton transfer at neutral pH. Other amino acid cofactors might also have been utilized, since directed evolution experiments have shown that RNA aptamers can be evolved to bind specifically to a number of amino acids (see, for example, Majerfeld et al 200529 and references therein).

However, the binding constants for amino acid: RNA complexes have proven to be quite weak, suggesting that covalent coupling might have had a greater impact on catalytic efficiency. Amino acids could have become directly attached to nucleotides through relatively simple chemistries30 and random insertion ofthese nucle-otides into catalysts would have led to at least some variants to show enhanced catalytic abilities. This would also have helped establish the selective pressures that would have led to the development of primordial amino acid biosynthetic pathways, translation and the inception ofthe genetic code, a hypothesis which has been proposed by Wong.31 However, in order to make sure this catalytic enhancement was inherited in each generation it would have been necessary to evolve some mechanism to attach a particular amino acid (or sets of closely related amino acids) to particular sites in a ribozyme. In this regard, Szathmary32 has suggested that amino acids with nucle-otide handles attached to them could have been used. This idea is analogous to the binding and use of nucleotide-based cofactors by catalysts. The nucleotide handle or adaptor would have provided a more convenient mechanism for the enzyme to specifically grab onto the amino acid cofactor. Indeed, with an oligonucleotide handle a cofactor could be directed to hybridize to a particular site within a ribozyme. In some ways, oligonucleotide-appended amino acids might have resembled the anti-codon stem loop of modern tRNAs and could have been the evolutionary step that led to encoded peptidation and the advent of the early genetic code.32

Assuming that there were sufficient and cogent evolutionary pressures for the invention oftranslation, then there is still the problem of how a huge, complex ribozyme like the ribosome could have been built up from simpler reaction mechanisms (as hypothesized by Woese as far back as the 1970s). Again, directed evolution can potentially provide experimental instantiation of what may have happened in the distant past, since relatively simple ribozymes can catalyze all of the chemistries needed for protein synthesis.

In modern translation, amino acids are polymerized into proteins by first making high-energy intermediates that then serve as substrates for encoding and ultimately peptide bond formation. High energy aminoacyl adenylates intermediates are synthesized from ATP by a class of enzymes called aminoacyl-tRNA synthetases (aaRS) and their cognate tRNAs (Fig. 11.4, Reaction 1). The aaRS then transfers the amino acid to its cognate tRNA via the formation of an ester between the carboxyl group on the amino acid and the 3' OH of its cognate tRNA to form an acylated tRNA (Fig. 11.4, Reaction 2). The acylated tRNA can then be used in protein synthesis via the peptidyl transferase activity of the ribosome (Fig. 11.4, Reaction 3). Since the ester linkage between the amino acid and the tRNA is a higher energy bond than the subsequent pep-tide bond this process is energetically favorable and should occur spontaneously when the two substrates are brought into proximity within the ribosome.

Satisfyingly, directed evolution experiments have shown that ri-bozymes are capable ofcatalyzing all ofthese steps. Work from Kumar and Yarus demonstrated that a ribozyme could be selected that could catalyze the formation ofan acyl-adenylate from ATP and carboxylic acids33 (Fig. 11.4, Reaction 1). The subsequent aminoacylation reaction has also been shown to be catalyzed by in vitro selected ribozymes (Fig. 11.4, Reaction 2). Yarus and coworkers again showed that an RNA could be selected that aminoacylated itself with phenylalanine (Phe) via phenylalanine adenylate as the starting material.34 Incredibly, one of the selected variants, RNA 77, could self-aminoacylate phenylalanine with a speed and specificity that exceeded tRNA amino-acylation by modern protein enzymes.35 Other directed evolution experiments have shown that selected ribozymes can also catalyze the transfer of other activated amino acids to their cognate tRNAs, a reaction identical to the second reaction catalyzed by the protein based aaRSs (see, for example, Lee et al36). The core catalytic activity ofthe self-aminoacylating RNA 77 eventually could be embodied in a minimal enzyme that was only 29 nucleotides long, though at reduced

Figure 11.4. Ribozyme catalysis of reactions involved in protein synthesis. Many of the biochemical reactions that occur during translation can also be catalyzed by ribozymes. 1) The activation of an amino acid to form a high energy 'activated' aminoacyl adenylate. 2) The transfer of the activated amino acid on to a hydroxyl on a RNA acceptor. The acceptor RNA in modern translation is tRNA, but in the RNA World could have been a ribozyme with a function similar to modern amino acyl tRNA synthetases. The attachment is indicated to be at either the 2' or 3' hydroxyl. 3) Two acylated amino acids can react to form a peptide bond. The peptide remains attached to the RNA, just as at the P site of the ribosome. Templating of this step has not been experimentally demonstrated, but such templating may have been the precursor of the modern genetic code. B is any nucleobase.

Figure 11.4. Ribozyme catalysis of reactions involved in protein synthesis. Many of the biochemical reactions that occur during translation can also be catalyzed by ribozymes. 1) The activation of an amino acid to form a high energy 'activated' aminoacyl adenylate. 2) The transfer of the activated amino acid on to a hydroxyl on a RNA acceptor. The acceptor RNA in modern translation is tRNA, but in the RNA World could have been a ribozyme with a function similar to modern amino acyl tRNA synthetases. The attachment is indicated to be at either the 2' or 3' hydroxyl. 3) Two acylated amino acids can react to form a peptide bond. The peptide remains attached to the RNA, just as at the P site of the ribosome. Templating of this step has not been experimentally demonstrated, but such templating may have been the precursor of the modern genetic code. B is any nucleobase.

rates compared to the longer variants.37 These results are significant in that they conclusively show that RNA is able to catalyze two of the three primary steps in translation with a catalyst that is a fraction ofcomplexity ofits modern counterparts, hinting that the molecular origins of translation could have arisen from relatively simple RNA catalysts and then evolved to greater complexity over time. The third step ofprotein synthesis can also be catalyzed by ribozymes (Fig. 11.4, Reaction 3). For example, the minimized RNA 77 could also catalyze the formation of a RNA-Phe-Phe dipeptide (Fig. 11.4, Reaction 3). Zhang and Cech have also selected ribozymes that have peptidyl transferase activity and can form peptides irrespective of the amino acids used.38 The next step in establishing an experimentally evolved translation apparatus will be to show sequence-specific decoding, something that should be relatively straightforward given that acy-lated amino acids can be tethered to oligonucleotides, as postulated by Szathmary.32 Nonetheless, future experiments will be needed to flesh out our understanding on this stage of evolution, perhaps using ribozymes that themselves template peptide formation, similar to the trans-acylation experiments already carried out by Lee et al.36

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