NMR investigation of RNA structure

It is fifty years since the first chemical synthesis of a dinucleoside phosphate and a dinucleotide with natural 3′→5′-internucleotide linkages was reported The main developments in the methodology of oligo- and poly-nucleotide synthesis that have taken place since are described

Oligo- and poly-nucleotides: 50 years of chemical synthesis

The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min-1) but not for cleavage, where docking (0.5 min-1) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G+1A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.

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Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer

The aminoacyl-tRNA synthetases are an ancient group of enzymes that catalyze the covalent attachment of an amino acid to its cognate transfer RNA. The question of specificity, that is, how each synthetase selects the correct individual or isoacceptor set of tRNAs for each amino acid, has been referred to as the second genetic code. A wealth of structural, biochemical, and genetic data on this subject has accumulated over the past 40 years. Although there are now crystal structures of sixteen of the twenty synthetases from various species, there are only a few high resolution structures of synthetases complexed with cognate tRNAs. Here we review briefly the structural information available for synthetases, and focus on the structural features of tRNA that may be used for recognition. Finally, we explore in detail the insights into specific recognition gained from classical and atomic group mutagenesis experiments performed with tRNAs, tRNA fragments, and small RNAs mimicking portions of tRNAs. © 2000 John Wiley & Sons, Inc. Biopoly 52: 1–28, 1999

Transfer RNA recognition by aminoacyl-tRNA synthetases.

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme.

Problems associated with the use of tetrabutylammonium fluoride like incomplete desilylation and removal of the tetrabutylammonium salts during large scale syntheses of oligoribonucleotides (RNA) have been eliminated by the use of triethylamine trihydrofluoride and precipitation of the RNA with 1-butanol. An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol. A new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides. RNA synthesized using these improvements has been shown to be biologically active by a comparative ribozyme-substrate assay.

Brian Sproat

Papers

An efficient method for the isolation and purification of oligoribonucleotides