Prebiotically plausible oligoribonucleotide ligation facilitated by chemoselective acetylation

The recent synthesis of pyrimidine ribonucleoside-2′,3′-cyclic phosphates under prebiotically plausible conditions has strengthened the case for the involvement of RNA at an early stage in the origin of life. However, a prebiotic conversion of these weakly activated monomers, and their purine counterparts, to the 3′,5′-linked RNA polymers of extant biochemistry has been lacking – previous attempts leading only to short oligomers with mixed linkages. Here we show that the 2′-hydroxyl group of oligoribonucleotide-3′-phosphates can be chemoselectively acetylated in water under prebiotically credible conditions, allowing rapid and efficient template-directed ligation. The 2′-O-acetyl group at the ligation junction of the product RNA strand can be removed under conditions that leave the internucleotide bonds intact. Remarkably, acetylation of mixed oligomers possessing either 2′- or 3′-terminal phosphates is selective for the 2′-hydroxyl group of the latter. This newly discovered chemistry thus suggests a prebiotic route from ribonucleoside-2′,3′-cyclic phosphates to predominantly 3′,5′-linked RNA via partially 2′-O-acetylated-RNA

Structural involvement in substrate recognition of an essential aspartate residue conserved in Mep/Amt and Rh-type ammonium transporters.

Ammonium transport proteins belonging to the Mep/Amt/Rh family are spread throughout all domains of life. A conserved aspartate residue plays a key role in the function of Escherichia coli AmtB. Here, we show that the analogous aspartate residue is critical for the transport function of eukaryotic family members as distant as the yeast transporter/sensor Mep2 and the human RhAG and RhCG proteins. In yeast Mep2, replacement of aspartate(186) with asparagine produced an inactive transporter localized at the cell surface, whilst replacement with alanine was accompanied by stacking of the protein in the endoplasmic reticulum. Introduction of an acidic residue, glutamate, produced a partially active protein. A carboxyl group at position 186 of Mep2 therefore appears mandatory for function. Kinetic analysis shows the Mep2(D186E) variant to be particularly affected at the level of substrate affinity, suggesting an involvement of aspartate(186) in ammonium recognition. Our data also put forward that ammonium recognition and/or transport by Mep2 is required for the sensor role played in the development of pseudohyphal growth. Finally, replacement of the conserved aspartate with asparagine in human RhAG and RhCG proteins resulted in the loss of bi-directional transport function. Hence, this aspartate residue might play a preserved functional role in Mep/Amt/Rh proteins.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe