Differential annotation of tRNA genes with anticodon CAT in bacterial genomes

We have developed three strategies to discriminate among the three types of tRNA genes with anticodon CAT (tRNA(Ile), elongator tRNA(Met) and initiator tRNA(fMet)) in bacterial genomes. With these strategies, we have classified the tRNA genes from 234 bacterial and several organellar genomes. These sequences, in an aligned or unaligned format, may be used for the identification and annotation of tRNA (CAT) genes in other genomes. The first strategy is based on the position of the problem sequences in a phenogram (a tree-like network), the second on the minimum average number of differences against the tRNA sequences of the three types and the third on the search for the highest score value against the profiles of the three types of tRNA genes. The species with the maximum number of tRNA(fMet) and tRNA(Met) was Photobacterium profundum, whereas the genome of one Escherichia coli strain presented the maximum number of tRNA(Ile) (CAT) genes. This last tRNA gene and tilS, encoding an RNA-modifying enzyme, are not essential in bacteria. The acquisition of a tRNA(Ile) (TAT) gene by Mycoplasma mobile has led to the loss of both the tRNA(Ile) (CAT) and the tilS genes. The new tRNA has appropriated the function of decoding AUA codons

Post-transcriptional nucleotide modification and alternative folding of RNA

Alternative foldings are an inherent property of RNA and a ubiquitous problem in scientific investigations. To a living organism, alternative foldings can be a blessing or a problem, and so nature has found both, ways to harness this property and ways to avoid the drawbacks. A simple and effective method employed by nature to avoid unwanted folding is the modulation of conformation space through post-transcriptional base modification. Modified nucleotides occur in almost all classes of natural RNAs in great chemical diversity. There are about 100 different base modifications known, which may perform a plethora of functions. The presumably most ancient and simple nucleotide modifications, such as methylations and uridine isomerization, are able to perform structural tasks on the most basic level, namely by blocking or reinforcing single base-pairs or even single hydrogen bonds in RNA. In this paper, functional, genomic and structural evidence on cases of folding space alteration by post-transcriptional modifications in native RNA are reviewed

Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns. Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases. The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end. In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities. Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining. Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing

Identification and Evolution of New Orthogonal Aminoacyl-tRNA Synthetase/tRNA Pairs for Genetic Code Expansion

Genetic code expansion is the branch of molecular biology aiming to expand the repertoire of amino acids which can be incorporated into proteins in vivo. A central challenge in expanding the genetic code of cells to incorporate non-canonical amino acids is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)–tRNA pairs (the components of the cellular translational machinery which specify the matching between codons and amino acids) that are orthogonal in their aminoacylation specificity. An orthogonal pair is composed of an aaRS which can interact with its partner tRNA, but not with any other tRNAs in the host, and a tRNA which is substrate to its partner aaRS, but not to any other aaRS in the host. In this research, candidate orthogonal tRNAs were identified from millions of sequences by implementing a computational analysis which scored their likelihood to be recognised by the endogenous aaRSs in E. coli, our model organism. I then developed a rapid, scalable new in vitro approach, named tRNA Extension (tREX), to determine the in vivo aminoacylation status of tRNAs. Using tREX, 243 candidate tRNAs were tested in E. coli and 71 orthogonal tRNAs were identified, covering 16 isoacceptor classes. 23 of those formed functional orthogonal tRNA–cognate aaRS pairs. By performing additional characterisation and molecular evolution of these newly identified functional pairs, we discovered 5 orthogonal pairs, 3 of which displayed high activity in amber suppression, the technique of choice used to implement genetic code expansion in model organisms. I additionally evolved new amino acid substrate specificities for two pairs. Finally, I use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expanded the number of orthogonal pairs available for genetic code expansion, provided a robust pipeline for the discovery of additional orthogonal pairs, and established a foundation for encoding the cellular synthesis of non-canonical biopolymers

Transfer RNA modification and infection – implications for pathogenicity and host responses

Open Access funded by the author(s).Transfer RNA (tRNA) molecules are sumptuously decorated with evolutionary conserved post-transcriptional nucleoside modifications that are essential for structural stability and ensure efficient protein translation. The tRNA modification levels change significantly in response to physiological stresses, altering translation in a number of ways. For instance, tRNA hypomodification leads to translational slowdown, disrupting protein homeostasis and reducing cellular fitness. This highlights the importance of proper tRNA modification as a determinant for maintaining cellular function and viability during stress. Furthermore, the expression of several microbial virulence factors is induced by changes in environmental conditions; a process where tRNA 2-thiolation is unequivocal for pathogenicity. In this review, we discuss the multifaceted implications of tRNA modification for infection by examining the roles of nucleoside modification in tRNA biology. Future development of novel methods and combinatory utilization of existing technologies will bring tRNA modification-mediated regulation of cellular immunity and pathogenicity to the limelight.Peer reviewe

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA(Ser) that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D–T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA

Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy

Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional (15N,1H)-HSQC spectra. In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids. At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible. Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected. Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction. Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and a-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain. The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor

Yeast aspartyl-tRNA synthetase residues interacting with tRNAAsp identity bases connectively contribute to tRNAAsp binding in the ground and transition-state complex and discriminate against non-cognate tRNAs

International audienceCrystallographic studies of the aspartyl-tRNA synthetase-tRNAAsp complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNAAsp. Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNAAsp recognition and acylation. The results showed that contacts with the acceptor A of tRNAAsp by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNAAsp. Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state. These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis. This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site. This signal might be conveyed via tRNAAspas suggested by the observed conformational change of this molecule upon interaction with AspRS. From binding free energy values corresponding to the different AspRS-tRNAAsp interaction domains, it might be concluded that upon complex formation, the anticodon interacts first. Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNAAsp and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs

RNA-dependent selenocysteine biosynthesis in eukaryotes and Archaea

Selenocysteine (Sec), the 21st genetically encoded amino acid, is the major metabolite of the micronutrient selenium. Sec is inserted into nascent proteins in response to a UGA codon. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNASec. While the formation of Sec-tRNASec from seryl-tRNASec by a single bacterial enzyme selenocysteine synthase (SelA) has been well described, the mechanism of Sec-tRNASec formation in archaea and eukaryotes remained poorly understood. Herein, biochemical and genetic data provide evidence that, in contrast to bacteria, eukaryotes and archaea utilize a different route to Sec-tRNASec that requires the tRNASec-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNA kinase (PSTK) first converts Ser-tRNASec to Sep-tRNASec. This misacylated tRNA is the obligatory precursor for a Sep-tRNA: Sec-tRNA synthase (SepSecS); this protein was previously annotated as Soluble Liver Antigen/Liver Pancreas (SLA/LP). SepSecS genes from Homo sapiens, the lower eukaryote Trypanosoma brucei and the archaea Methanocaldococcus jannaschii and Methanococcus maripaludis complement an Escherichia coli DeltaselA deletion strain in vivo. Furthermore, genetic analysis of selenoprotein biosynthesis in T. brucei in vivo demonstrated that eukaryotes have a single pathway to Sec-tRNASec that requires Sep-tRNASec as an intermediate. Finally, purified recombinant SepSecS converts Sep-tRNA Sec into Sec-tRNASec in vitro in the presence of sodium selenite and purified E. coli selenophosphate synthetase.The final step in Sec biosynthesis was further investigated by a structure-based mutational analysis of the M. maripaludis SepSecS and by determining the crystal structure of human SepSecS complexed with tRNA Sec, phosphoserine and thiophosphate at 2.8 A resolution. In vivo and in vitro enzyme assays support a mechanism of Sec-tRNASec formation based on pyridoxal phosphate, while the lack of active site cysteines demonstrates that a perselenide intermediate is not involved in SepSecS-catalyzed Sec formation. Two tRNASec molecules, with a fold distinct from other canonical tRNAs, bind to each human SepSecS tetramer through their unique 13 base-pair acceptor-TPsiC arm. The tRNA binding induces a conformational change in the enzyme\u27s active site that allows a Sep covalently attached to tRNASec, but not free Sep, to be oriented properly for the reaction to occur

Structural analysis of stalled ribosomal complexes and their respective rescue mechanisms by Cryo-Electron Microscopy

The ribosome is a multifunctional ribonucleoprotein complex responsible for the translation of the genetic code into proteins. It consists of two subunits, the small ribosomal subunit and the large ribosomal subunit. During initiation of translation, both subunits join and form a functional 70S ribosome that is capable of protein synthesis. In the course of elongation, the ribosome synthesizes proteins according to the codons on the mRNA until it encounters a stop codon leading to the recruitment of release factors 1 or 2 followed by release of the nascent chain. Upon release of the polypeptide chain the subunits dissociate from each other and can be recruited for another round of translation. There are two scenarios that interfere with active translation, namely the formation of so called ‘non-stop’ or ‘no-go’ complexes. In both cases, ribosomes pause translation and without interference of additional factors, they would become stalled. Accumulation of such events leads to a decrease of ribosomal subunits that can be recruited for translation, ultimately resulting in the death of the cell. Using cryo-electron microscopy (cryo-EM), we obtained the structure of alternative rescue factor A (ArfA) together with release factor 2 bound to a ‘non-stop’ complex. Our reconstructions showed that the C-terminal domain of ArfA occupies the empty mRNA channel on the SSU, whereas the N-terminal domain provides a platform for recruiting RF2 in a stop codon-independent way. Thereby, ArfA stabilizes a unique conformation of the switch loop of RF2, responsible for directing the catalytically important GGQ motif towards the PTC. The high-resolution structure of ArfA allowed us to compare its mode of action with trans-translation and alternative rescue factor B, two other factors operating on ‘non-stop’ complexes. A second project focused on elongation factor P (EF-P), a factor that alleviates stalling on polyproline stalled ribosomes. Applying cryo-EM, we were able to show that in the absence of EF-P, the nascent chain is destabilized as the polyproline moiety attached to the P-tRNA is not able to accommodate within the ribosomal tunnel. Binding of modified EF-P to the polyproline stalled complex stabilizes the P-site tRNA and especially the CCA, thereby forcing the nascent chain to adopt an alternative conformation that is favorable for translation to proceed