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.     

Native transfer RNA catalyzes Diels-Alder reaction

In this paper we show that transfer ribonucleic acids (tRNAs) catalyze the Diels-Alder cycloaddition reaction. A new DNA oxidative damage product, 6-furfuryladenine (kinetin) or its riboside (diene), was transformed with dimethyl acetylenedicarboxylate or maleic anhydride (dienophile). The reaction proceeds in the presence of tRNA at high pressure but not at ambient condition. If so tRNA in prebiotic conditions (RNA world) had at least two functions: catalytic and a carrier of genetic information. It means that tRNA at high pressure shows catalytic properties and is a true Diels-Alderase.     

Structure and Function of tmRNA (10Sa RNA)

Modern data on the structure and function of transport/messenger (tm) RNA are reviewed. This stable RNA is involved in releasing ribosomes that are unable to complete protein synthesis on mRNA lacking the stop codon. The resulting abnormal proteins are rapidly degraded by specific proteases, which recognize a signal peptide encoded by the template region of tmRNA. The discovery of trans-translation has caused a particular interest in structural and functional studies of tmRNA.     

Effect of seed germination on levels of tRNA aminoacylation

In ungerminated seeds of Lupinus luteustRNAs are aminoacylated 10% or less depending on species of tRNA. The levels of tRNA aminoacylation for specific tRNAs increase steadily during seed germination. Specific tRNAs in cotyledons and axes of 3-day-old seedlings are aminoacylated to a similar extent. No significant changes are observed in the tRNA population during germination.     

Statistical evidence for remnants of the primordial code in the acceptor stem of prokaryotic transfer RNA

Summary The specificity of interaction of amino acids with triplets in the acceptor helix stem of tRNA was investigated by means of a statistical analysis of 1400 tRNA sequences. The imprint of a prototypic genetic code at position 3–5 of the acceptor helix was detected, but only for those major amino acids, glycine, alanine, aspartic acid, and valine, that are formed by spark discharges of simple gases in the laboratory. Although remnants of the code at position 3–5 are typical for tRNAs of archaebacteria, eubacteria, and chloroplasts, eukaryotes do not seem to contain this code, and mitochondria take up an intermediary position. A duplication mechanism for the transposition of the original 3–5 code toward its present position in the anticodon stern of tRNA is proposed. From this viewpoint, the mode of evolution of mRNA and functional ribosomes becomes more understandable.Offprint requests to: W. Moller     

E292 is important for the aminoacylation activity of Escherichia coli leucyl-tRNA synthetase

Escherichia coli leucyl-tRNA synthetase (LeuRS) has a large connecting polypeptide (CP1) inserted into its active site. It was demonstrated that the peptide bond between E292–A293 was crucial for the aminoacylation activity of E. coli LeuRS. To investigate the effect of E292 on the function of Escherichia coli LeuRS, E292 was mutated to K, F, S, D, Q and A. These mutations at 292 did not change the specific activity of the amino acid activation reaction. Though the conformational change of these mutants was not detected in CD, their aminoacylation activities were impaired to varying extents. The mutation of E to K decreased the aminoacylation activity to the largest extent. Analysis of the K_m values of these mutants for the three substrates showed that the E292 was not involved in the binding of leucine and that all mutants had stronger binding with ATP.     

1964: The first model for the shape of a transfer RNA molecule. An account of an unpublished small-angle X-ray scattering study

The shape of non-fractionated Escherichia coli transfer RNA molecules in solution was investigated using small-angle X-ray scattering during the years 1960-1962 at the Centre de Recherche sur les Macromolécules in Strasbourg. The innermost region of the scattering curve yielded the average molecular weight (Mr) and the radius of gyration (Rg) of the particles, whereas the experimental data at large angles could be approximated at best by the scattering curve of a kinked rod-shaped molecule. The simplest model that was compatible with Mr, Rg, and the mass per unit length of the rod was a boomerang-shaped particle made of two double helical stems connected by a sharp kink. This model that eventually proved similar to the high-resolution L-shaped structure, was presented in my Ph.D. dissertation (J. Witz, Etude de la structure de quelques polynucléotides en solution par diffusion centrale des rayons X, Ph.D. dissertation, University of Strasbourg, France, 1964) but has never been published in detail. It is the purpose of this note to recall this story.     

Molecular evolution of transfer RNA from two precursor hairpins: Implications for the origin of protein synthesis

In this paper we are going to present a model for the coevolution of major components of the protein synthesis machinery in a primordial RNA world. We propose that the essential prerequisites for RNA-based protein synthesis, i.e., tRNA-like molecules, ribozymic charging catalysts, small-subunit(SSU) rRNA, and large-subunit(LSU) rRNA, evolved from the same ancestral RNA molecule. Several arguments are considered which suggest that tRNA-like molecules were derived by tandem joining of template-flanking hairpin structures involved in replication control. It is further argued that the ancestors of contemporary group I tRNA introns catalyzed such hairpin joining reactions, themselves also giving rise to the ribosomal RNAs. Our model includes a general stereochemical principle for the interaction between ribozymes and hairpin-derived recognition structures, which can be applied to such seemingly different processes as RNA polymerization, aminoacylation, tRNA decoding, and peptidyl transfer, implicating a common origin for these fundamental functions. These and other considerations suggest that generation and evolution of tRNA were coupled to the evolution of synthetases, ribosomal RNAs, and introns from the beginning and have been a consequence arising from the original function of tRNA precursor hairpins as replication and recombination control elements. Correspondence to: T.P. Dick     

Qualitative and quantitative variation of tRNA in certain invertebrates

Total tRNA was isolated, purified and quantitated from earthworm, cockroach, fresh water mussel and rat liver. The total tRNA content of invertebrates was found to be much lower than that of rat liver. When checked for aminoacylation capacity with homologous and heterologous enzymes and algal protein hydrolysate, the tRNA preparation from rat liver and fresh water mussel, a mollusc, were found to be active. On the other hand, the tRNAs from earthworm, an annelid, and cockroach, an arthropod, were completely inactive with the homologous enzymes but showed partial activity with heterologous enzymes. Similar results were obtained with individual amino acids also. The low activity or inactivity of earthworm and cockroach tRNAs appears to be due to certain endogenous aminoacylation inhibitors. This work was carried out at the School of Life Sciences, University of Hyderabad, Hyderabad 500 134, India.     

Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea

cis-Zeatin riboside was identified in transfer-RNA hydrolysates from both normal and crown-gall tissue of Vinca rosea L. The trans-isomer was associated exclusively with the crowngall transfer-RNA. The importance of these observations is discussed in relation to biosynthesis of free cytokinins.Abbreviations GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - TMSi trimethylsilyl - tRNA transfer RNA - ZR zeatin riboside     

An RNA replisome as the ancestor of the ribosome

Summary The ribosome is proposed to have evolved from an earlier RNA-replisome, which synthesized RNA. Ancestral tRNA molecules originally were loaded with trinucleotide sequences and donated them to growing RNA chains. The enzymatic addition of the C-C-A trinucleotide to presentday transfer RNA molecules is a carryover from this function. The strategies of reading RNA sequences by triplet codons and of housing information genetically in special repository molecules predates the origin of protein and DNA. These latter two polymers arose together at the time when the RNA replisome was converted to a ribosome.     

Structural effects of modified ribonucleotides and magnesium in transfer RNAs

Modified nucleotides are ubiquitous and important to tRNA structure and function. To understand their effect on tRNA conformation, we performed a series of molecular dynamics simulations on yeast tRNA^Phe and tRNA_init, Escherichia coli tRNA_init and HIV tRNA^Lys. Simulations were performed with the wild type modified nucleotides, using the recently developed CHARMM compatible force field parameter set for modified nucleotides (J. Comput. Chem. 2016, 37, 896), or with the corresponding unmodified nucleotides, and in the presence or absence of Mg²⁺. Results showed a stabilizing effect associated with the presence of the modifications and Mg²⁺ for some important positions, such as modified guanosine in position 37 and dihydrouridines in 16/17 including both structural properties and base interactions. Some other modifications were also found to make subtle contributions to the structural properties of local domains. While we were not able to investigate the effect of adenosine 37 in tRNA_init and limitations were observed in the conformation of E. coli tRNA_init, the presence of the modified nucleotides and of Mg²⁺ better maintained the structural features and base interactions of the tRNA systems than in their absence indicating the utility of incorporating the modified nucleotides in simulations of tRNA and other RNAs.