In modern organisms, three main reactions enable the conversion of the information contained in genes into functional proteins through the rules of the genetic code. The first reaction (1) corresponds to the formation of aa-AMP from of an amino acid (aa) and an ATP molecule, and is a thermodynamic requirement for the second reaction (2), the attachment of the amino acid onto the 3 end of a cognate transfer RNA (tRNA). The third reaction (3) leads to the polymerization of tRNA-bound amino acids into proteins during the translation process (on the ribosome). Approximately twenty versions of the aminoacyl-tRNA synthetase are capable of successively catalyzing reactions 1 and 2. The early genetic system could not possibly have relied on any such coded proteins, and it is therefore thought that some RNA molecules were able to catalyze these reactions following the rules of a simplified genetic code. The goal of this project is to clarify the mechanisms of interaction among RNA structures, ATP and amino acids (as well as AMP-activated amino acids) by which these three main chemical reactions could be fulfilled without protein. Studies have already demonstrated that reaction 2 and an analog of reaction 1 can be catalyzed by certain RNAs. The product of reaction 1 (aa-AMP) is however quickly hydrolyzed in water, proving difficult to study. This instability requires the coupling of reactions 1 and 2 on a same molecule, and occurs precisely on the aminoacyl-tRNA synthetases. It is therefore expected that some yet unidentified RNAs have this same catalytic property. The resolution of this issue constitutes the basis of the present research project. Data from earlier experimental studies, suggest mechanisms by which this coupling can be achieved on very short RNAs (~25 bases) that form simple folding structures, such as small bulges. The critical parameters allowing these reactions will be studied both experimentally and computationally (with molecular dynamic simulations). The PIs will also use selection-amplification procedures (SELEX) to uncover these RNAs from pools of random molecules. Reaction 3 will also be investigated, in particular to establish whether the ribosome is indispensable for peptide bond formation, or if it is only necessary to establish a ratchet-like mechanism. This work will clarify the implications of the elementary properties of the amino acids and the nucleotides in water for the mechanisms responsible for the emergence of the genetic code. Since the polymerization of the amino acids through reactions 1-3 is a process out of equilibrium, new results in this field will help to clarify the connection between biological coding and the laws of non-equilibrium thermodynamics. This project will be beneficial to undergraduate students, introducing them to one of most fundamental issues of modern biophysics, the bridge between the work of Boltzmann (thermodynamics) and Turing (coding).
It was observed in the 80’s that, deep in the ocean, along the ridges separating tectonic plates, there are thermal vents that inject enriched water at temperatures of up to 400? into an ocean with a surrounding temperature 5?. Those vents are of meter length scale with a large number of pores in the vents ranging in size from microns to millimeters. Thus, small length scale temperature gradients in the pores are part of natural phenomena. It has been suggested that thermal vents could be the location where life originates, given the presence of seawater, various chemical compounds emitted by small volcanoes, the high temperature of the emitted fluid with cold fluid around and also complete isolation from devastating UV solar radiation. Following on such hypothesis, and assuming an RNA world at the origin of life, we performed many experiments to test the effect of temperature gradients on DNA and RNA and showed that accumulation and amplification of DNA is possible. In this grant proposal, we revisited DNA and RNA in a temperature gradient and in the presence of a large volume fraction of another polymer. Focusing an infrared laser beam to induce local heating, the polymer moves away, resulting in an exponential concentration of the polymer. This creates an entropic potential well in which DNA or RNA polymers localize themselves, depending on their length. We have alas studied thermal separation of RNA whose size varies from 100 base pairs (as small as transfer RNA) to a few 1000 base pairs (as long as messenger RNA). Small RNA were discovered to have a wide range of functions. In those small functional RNA, the stem-loop structure is the basic motif for RNA folding with a rigid stem (50 nm persistence length) and a flexible loop. We find that the selection of RNA depends on the total length of their stems. Hence this motif selection induces some sequence selection. A consequence of this effect concerns the origin of life hypothesis near thermal vents where large temperature gradients may select small RNA in the pore present in the solid deposits. The conclusion is that, in the pores localized in the small volcano forming an hydrothermal vent, DNA can be concentrated, amplified, size selected, and that small RNA could be also somewhat sequence selected. But as hydrothermal vents are located deep in the ocean, an important question is how proteins can sustain the harsh pressure condition of up to 1000 Atmospheres (10 kilometers in depth in the ocean). In this grant proposal we have studied at what pressure and temperature a protein like RecA loses its function and we found that up to 700 Atmospheres the protein conserves its function, implying that pressure is not too severe a perturbation for life forms.
