12-19-2002, 11:07 AM
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<a href="http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v420/n6917/full/nature01185_fs.html" target="_blank">Nature 420, 841 - 844 (2002); doi:10.1038/nature01185</a>
A ribozyme composed of only two different nucleotides
JOHN S. READER AND GERALD F. JOYCE
Quote:
A good starting point for the evolution of a catalyst that contains only two different subunits was the R3 ligase ribozyme, which contains only adenine, guanine and uracil nucleotides8, 9. This ribozyme catalyses attack of the 3'-hydroxyl of an RNA substrate on the 5'-triphosphate of the ribozyme, forming a 3',5'-phosphodiester and releasing inorganic pyrophosphate. The chemistry of this reaction is identical to that catalysed by modern RNA polymerase proteins. A templating region within the ribozyme is responsible for binding the RNA substrate, and the sequences of both the template and substrate can be designed such that they contain only adenine and uracil residues. In that format, both the ribozyme and substrate are completely devoid of cytosine and undergo RNA ligation with a catalytic rate, kcat, of 0.013 min-1 and Michaelis constant, Km, of 6.2 µM (ref. 9).
The R3 ribozyme was found to be highly tolerant of base substitutions involving replacement of every adenine by 2,6-diaminopurine (D). Three bonds are formed between 2,6-D and uracil in the context of a Waston–Crick base pair10, in comparison with adenine, which forms only two. Despite this difference, the D-substituted R3 ligase (Fig. 1a) retained a kcat of 0.001 min-1 and Km of 12 µM, and reacted to a maximum extent of about 40%. Substituting diaminopurine 5'-triphosphate (DTP) for ATP when transcribing AT-containing DNA templates was found to have two important advantages for in vitro evolution experiments. First, this substitution reduced 'slippage' of the RNA polymerase11 as it proceeded along the DNA template. Second, transcription could be initiated with a D residue at the 5' end of the RNA, allowing transcripts to be produced in the complete absence of GTP and CTP. For all these reasons, the R3 ligase, modified to contain D, G and U, was chosen as the starting point for the evolution of ligase ribozymes that contain only D and U.
[...]
It has been suggested that the original genetic system contained only two different nucleotides3-7, and subsequently evolved to its present, more complex form. A binary genetic system may have been advantageous during the early history of life on earth when the availability of all four nucleotides might have been difficult to maintain. Cytosine nucleotides are especially problematic because they undergo rapid deamination to uridylate, with a half-life of 19 days at pH 7 and 100 °C (ref. 2). Thus the GC pairing may not have been sustainable until the invention of a mechanism for restoring cytosine to uracil. By comparison, the half-life of adenine or diaminopurine at pH 7 and 100 °C is about one or two years, respectively2. The prebiotic synthesis of adenine or diaminopurine proceeds with comparable efficiency, both compounds being obtained in good yield starting from aqueous ammonium cyanide14, 15.
Nucleic acid enzymes composed of only D and U residues pay a heavy price for their simplified composition in terms of both catalytic rate and the fraction of molecules that are in an active conformation. Nonetheless, darwinian evolution can produce catalytically active structures even from such a severely restricted chemical repertoire. The conformational plasticity of DU-containing RNA may even offer an advantage with regard to the ability to explore multiple structures for a given sequence16.
Ribozymes composed of other pairs of nucleotides, such as A and U, G and C, or even A and I (inosine), may be possible. It seems less likely that polymers composed of only two amino acids could exhibit appreciable catalytic activity. Thus far a minimum of 14 amino acids has been used to construct a catalytic polypeptide17, and as few as seven have been shown to be necessary to define a folded tertiary structure18. The absolute minimum number of distinct subunits that could be used to construct a functional informational macromolecule is two, as was the case in this study. Without at least two different subunits, there is no information and thus no basis for darwinian evolution.
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[ December 19, 2002: Message edited by: Principia ]</p>
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