Brain transfer RNA

Transfer RNAs were isolated from rat and calf brains and their nucleosides were analysed by tritium derivative technique. Qualitative changes in the minor nucleoside components were compared on the fluorograms which showed differences in the intensities of spots. Cerebellar and cortical tRNAs were also compared, but revealed no significant quantitative differences in their methylated constituants despite 60% higher methyltransferase activity observed in cerebellum compared to cerebral cortex. An overall similarity was noticed between the relative proportions of the major and minor nucleosides of tRNAs derived from rat or calf brain, expressed as mol %. Brain tRNA was also analysed by two-dimensional polyacrylamide gel electrophoresis which showed qualitative and quantitative changes during postnatal development.     

Phylogeny of transfer RNA

Phylogenetic trees of transfer RNA specific for phenylalanine, methionine initiator glycine and valine are constructed. Although the exact relationships between taxa cannot be obtained from the mere analysis of the sequences some conclusions can be drawn about the evolution of this molecule.     

Phylogeny of transfer RNA

Phylogenetic trees of transfer RNA specific for phenylalanine, methionine initiator glycine and valine are constructed. Although the exact relationships between taxa cannot be obtained from the mere analysis of the sequences some conclusions can be drawn about the evolution of this molecule.This research was supported by DAAD (Germany) and Comité de Investigaciones de la Universidad de los Andes Bogotá, Columbia. Dedicated to my Son Andres Felipe.     

Evolution of transfer RNA

Evolution by gene duplication and subsequent divergence is indicated by similarities common to 43 different transfer RNAs. Pairwise comparisons of these tRNAs reveal additional similarity, greatest for certain pairs of tRNAs for the same amino acid in the same organism, and also occurring in certain pairs of tRNAs for different amino acids in the same organism. Although tRNAs functionally interact with several other molecules, there have been surprisingly few restrictions on the divergence of their primary structures. This divergence has proceeded so far that clear phylogenetic separations are absent in most cases: it it impossible to construct a coherent phylogeny for most of the 43. Selection and stochastic processes have both been active in the evolution of tRNA. Selection has favored moderate change more than expected and has reduced radical change below that expected from stochastic processes alone. Two obvious effects of selection are nine invariant loci, another five that are always purines and five others that are always pyrimidines, in the tRNAs involved in protein synthesis. In addition to these constraints in the primary nucleotide sequence, the method of “identical site equivalents”, introduced here, demonstrates that further constraints exist equivalent to about 12 additional invariant loci. These “invisible” restraints reflect disperse chemical forces maintaining the tertiary structure and reducing evolutionary divergence to an extent quantitatively comparable to that of the nine observable invariant loci. The average divergence (49·4%) for pairs of tRNAs for different amino acids involved in protein synthesis represents an equilibrium between natural selection and stochastic processes. These tRNAs have had time to diverge nearly to the 75% maximum expected from stochastic process alone; this is shown by comparing the two glycine tRNAs involved in peptidoglycan synthesis with tRNAs for different amino acids participating in polypeptide synthesis. The rates of nucleotide replacements in genes coding for the tRNAs and the cytochromes c are about the same: 2 × 10 ^?10 replacements per nucleotide site per year.     

Photobinding of 8-methoxypsoralen to transfer RNA and 5-fluorouracil-enriched transfer RNA.

The photobinding of [3H]8MOP to tRNA upon irradiation at 365 nm in the absence of O2 was determined by gel filtration. The maximum photobinding was found to be ca. 4 mol of 8MOP er mol of tRNA and 5FU-tRNA, with an overall quantum yield of 2.3 X 10(-3). The photobinding kinetics for 8MOP-tRNA showed an apparent induction period or sigmoidal kinetic curve, indicating a specific initial photobinding site on tRNA which was identified as 4-thiouridine at position 8 from the 5'-end of Escherichia coli tRNA. Photobinding of 8MOP to 5FU-tRNA proceeded without an apparent induction period. 8MOP-tRNA and 8MOP-5FU-tRNA adducts were characterized by absorption, fluorescence, and CD spectroscopy. A modified procedure was also developed to analyze the nucleoside composition in modified 8MOP-tRNA and 8MOP-5FU-tRNA. The results showed that 8MOP photochemically added mainly to pyrimidine bases. The photobinding of 8MOP changed the conformation (secondary in particular) of tRNA and inhibited aminoacyl-tRNA synthetase activity.     

Differences between transfer RNA molecules

Computer-assisted comparisons of 67 tRNA sequences that function in Escherichia coli or Salmonella typhimurium were used to identify single and multiple nucleotide positions that maximally distinguish the 20 amino acid acceptor groups. Positions in the anticodon were identified most frequently, as expected from the decoding function of this region of the tRNA. The biological function, if any, of positions outside the anticodon may include specificity for aminoacyl-tRNA synthetase enzymes.     

RNA quality control: degradation of defective transfer RNA

The distinction between stable (tRNA and rRNA) and unstable (mRNA) RNA has been considered an important feature of bacterial RNA metabolism. One factor thought to contribute to the difference between these RNA populations is polyadenylation, which promotes degradation of unstable RNA. However, the recent discovery that polyadenylation also occurs on stable RNA led us to examine whether poly(A) might serve as a signal for eliminating defective stable RNAs, and thus play a role in RNA quality control. Here we show that a readily denaturable, mutant tRNA(Trp) does not accumulate to normal levels in Escherichia coli because its precursor is rapidly degraded. Degradation is largely dependent on polyadenylation of the precursor by poly(A) polymerase and on its removal by polynucleotide phosphorylase. Thus, in the absence of these two enzymes large amounts of tRNA(Trp) precursor accumulate. We propose that defective stable RNA precursors that are poorly converted to their mature forms may be polyadenylated and subsequently degraded. These data indicate that quality control of stable RNA metabolism in many ways resembles normal turnover of unstable RNA.     

The RNA origin of transfer RNA aminoacylation and beyond

Aminoacylation of tRNA is an essential event in the translation system. Although in the modern system protein enzymes play the sole role in tRNA aminoacylation, in the primitive translation system RNA molecules could have catalysed aminoacylation onto tRNA or tRNA-like molecules. Even though such RNA enzymes so far are not identified from known organisms, in vitro selection has generated such RNA catalysts from a pool of random RNA sequences. Among them, a set of RNA sequences, referred to as flexizymes (Fxs), discovered in our laboratory are able to charge amino acids onto tRNAs. Significantly, Fxs allow us to charge a wide variety of amino acids, including those that are non-proteinogenic, onto tRNAs bearing any desired anticodons, and thus enable us to reprogramme the genetic code at our will. This article summarizes the evolutionary history of Fxs and also the most recent advances in manipulating a translation system by integration with Fxs.