ASGSB 1998 Annual Meeting Abstracts

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PRIMORDIAL PEPTIDE SYNTHESIS IN THE ABSENCE OF A TEMPLATE OR RIBOSOME.   D.C. Larkin¹, S.A. Martinis¹, D.J. Roberts², and G.E. Fox¹. ¹Dept of Biology & Biochemistry and ²Civil & Environmental Engineering, University of Houston.

In order to comprehend the origin of life on Earth, it is essential to understand the genesis of the complex protein synthesis machinery shared by all extant organisms. A promising model envisions co-evolution of this translation machinery from a small RNA/peptide complex capable of peptide synthesis to the modern form of template-directed protein synthesis. Can such an RNA/peptide complex actually catalyze the formation of a peptide bond? We are attempting to determine the feasibility of such a scenario. To this end, we have shown that leu-leu dipeptides are formed in the presence of a specific catalyst (ala-his), and aminoacylated tRNA with or without its anticodon domain. The mechanism of this apparent catalytic behavior was analyzed using specific molecule analogues with structural similarity to ala-his and a series of engineered RNA constructs. The next step will be to determine if the synthetic capability is maintained for RNAs of less than 25 nucleotides.

RNAs derived from leucine tRNA were made by in vitro run-off transcription from specific DNA templates using T7 RNA polymerase and aminoacylated with Leu-tRNA synthetase. Aminoacylated RNAs were purified under acidic conditions and incubated with potential dipeptide catalysts in a range of acidic to basic conditions. A thin layer chromatography assay for separating and purifying the leu-leu product was developed. Product identity was established based on mobility using phosphoroimage visualization and confirmed by mass spectroscopy. (Supported by NASA: NAG5-4004 and NGT5-50182).

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