Aminoacylation of RNA minihelices with alanine

The genetic code is determined by both the specificity of the triplet anticodon of tRNAs for codons in mRNAs and the specificity with which tRNAs are charged with amino acids. The latter depends on interactions between tRNAs and their charging enzymes, and an advance in understanding such interactions was provided recently by the demonstration that a major determinant of the identity of alanine tRNA is located in the amino-acid acceptor helix. Multiple substitutions in many distinct parts of the molecule do not prevent aminoacylation with alanine. Substitution of the G3.U70 base pair with G3.C70 or A3.U70 in the acceptor helix prevents aminoacylation in vivo and in vitro, however, and the introduction of this base pair into tRNA(Cys) (ref. 1) or tRNA(Phe) (refs 1, 2) enables both to accept alanine. The importance of a single base pair in the acceptor helix and the results of recent footprinting experiments promoted us to investigate the possibility that a minihelix, composed only of the amino-acid acceptor-T psi C helix, could be a substrate for alanine tRNA synthetase. We show here that a synthetic hairpin minihelix can be enzymatically aminoacylated with alanine. Alanine incorporation requires a single G.U base pair, and occurs in helices that otherwise differ significantly in sequence. Aminoacylation can be achieved with only seven base pairs in the helix.     

Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration

Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.     

Functional contacts of a transfer RNA synthetase with 2'-hydroxyl groups in the RNA minor groove.

The functional analysis of determinants on RNA has been largely limited to molecules that contain naturally occurring ribonucleotides, so little is known about the role of 2'-hydroxyl groups in protein-RNA recognition. A single base pair (G3.U70) in the acceptor stem of tRNA(Ala) is the principal element for specific recognition by Escherichia coli alanine-tRNA synthetase. This tRNA synthetase aminoacylates small RNA helices that contain the G3.U70 base pair. Furthermore, removal of the G3 exocyclic 2-amino group that projects into the minor groove eliminates aminoacylation. This 2-amino group is flanked on either side by ribose 2'-hydroxyl groups that line the minor groove. Here we use chemical synthesis to construct 32 helices that make deoxy and O-methyl substitutions of individual and multiple 2'-hydroxyl groups near and beyond the G3.U70 base pair and find that functional 2'-hydroxyl contacts are clustered within a few ?ngstroms of the critical 2-amino group. These contacts are highly specific and make a thermodynamically significant contribution to RNA recognition.     

Anticodon and acceptor stem nucleotides in tRNA(Gln) are major recognition elements for E. coli glutaminyl-tRNA synthetase

The correct attachment of amino acids to their corresponding (cognate) transfer RNA catalysed by aminoacyl-tRNA synthetases is a key factor in ensuring the fidelity of protein biosynthesis. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system to study this highly specific recognition process, also referred to as 'tRNA identity'. Accurate acylation of tRNA depends mainly on two principles: a set of nucleotides in the tRNA molecule (identity elements) responsible for proper discrimination by aminoacyl-tRNA synthetases and competition between different synthetases for tRNAs. Elements of glutamine identity are located in the anticodon and in the acceptor stem region, including the discriminator base. We report here the production of more than 20 tRNA(2Gln) mutants at positions likely to be involved in tRNA discrimination by the enzyme. Unmodified tRNA, containing the wild-type anticodon and U or G at its 5'-terminus, can be aminocylated by GlnRS with similar kinetic parameters to native tRNA(2Gln). By in vitro aminoacylation the mutant tRNAs showed decreases of up to 3 x 10(5)-fold in the specificity constant (kcat/KM)14 with the major contribution of kcat. Despite these large changes, some of these mutant tRNAs are efficient amber suppressors in vivo. Our results show that strong elements for glutamine identity reside in the anticodon region and in positions 2 and 3 of the acceptor stem, and that the contribution of different identity elements to the overall discrimination varies significantly. We discuss our data in the light of the crystal structure of the GlnRS:tRNA(Gln) complex.     

Protein biosynthesis in organelles requires misaminoacylation of tRNA

In the course of our studies on transfer RNA involvement in chlorophyll biosynthesis, we have determined the structure of chloroplast glutamate tRNA species. Barley chloroplasts contain in addition to a tRNA(Glu) species at least two other glutamate-accepting tRNAs. We now show that the sequences of these tRNAs differ significantly: they are differentially modified forms of tRNA(Gln) (as judged by their UUG anticodon). These mischarged Glu-tRNA(Gln) species can be converted in crude chloroplast extracts to Gln-tRNA(Gln). This reaction requires a specific amidotransferase and glutamine or asparagine as amide donors. Aminoacylation studies show that chloroplasts, plant and animal mitochondria, as well as cyanobacteria, lack any detectable glutaminyl-tRNA synthetase activity. Therefore, the requirement for glutamine in protein synthesis in these cells and organelles is provided by the conversion of glutamate attached to an 'incorrectly' charged tRNA. A similar situation has been described for several species of Gram-positive bacteria. Thus, it appears that the occurrence of this pathway of Gln-tRNA(Gln) formation is widespread among organisms and is a function conserved during evolution. These findings raise questions about the origin of organelles and about the evolution of the mechanisms maintaining accuracy in protein biosynthesis.     

Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme

In modern organisms, protein enzymes are solely responsible for the aminoacylation of transfer RNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalysing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated l-phenylalanine derivatives. In addition to a more than 10(5) rate enhancement and more than 10(4)-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3'-OH. Here we report the 2.8-A resolution structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA molecule, flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallographically independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases to increase specificity.     

A new understanding of the decoding principle on the ribosome

During protein synthesis, the ribosome accurately selects transfer RNAs (tRNAs) in accordance with the messenger RNA (mRNA) triplet in the decoding centre. tRNA selection is initiated by elongation factor Tu, which delivers tRNA to the aminoacyl tRNA-binding site (A site) and hydrolyses GTP upon establishing codon-anticodon interactions in the decoding centre. At the following proofreading step the ribosome re-examines the tRNA and rejects it if it does not match the A codon. It was suggested that universally conserved G530, A1492 and A1493 of 16S ribosomal RNA, critical for tRNA binding in the A site, actively monitor cognate tRNA, and that recognition of the correct codon-anticodon duplex induces an overall ribosome conformational change (domain closure). Here we propose an integrated mechanism for decoding based on six X-ray structures of the 70S ribosome determined at 3.1-3.4?? resolution, modelling cognate or near-cognate states of the decoding centre at the proofreading step. We show that the 30S subunit undergoes an identical domain closure upon binding of either cognate or near-cognate tRNA. This conformational change of the 30S subunit forms a decoding centre that constrains the mRNA in such a way that the first two nucleotides of the A codon are limited to form Watson-Crick base pairs. When U·G and G·U mismatches, generally considered to form wobble base pairs, are at the first or second codon-anticodon position, the decoding centre forces this pair to adopt the geometry close to that of a canonical C·G pair. This by itself, or with distortions in the codon-anticodon mini-helix and the anticodon loop, causes the near-cognate tRNA to dissociate from the ribosome.     

Species-specific aminoacylation of Oryza sativa mitochondrial tRNA^Trp

Accuracy in protein synthesis is essential to the survival of all organisms. Aminoacyl-tRNA synthetases catalyzed aminoacylation is the basis of the fidelity of protein synthesis[1]. In recent years, the identification of nucleotide determinants, specific…     

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A?site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A?site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G-ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G-ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P?site) on the 30S head and simultaneously establishes interaction with the exit site (E?site) on the 30S platform, a novel intra-subunit 'pe/E' hybrid state is formed. This state is stabilized by domain?IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the 'missing link' in terms of tRNA intermediates involved in the universally conserved translocation process.     

An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes

Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs. There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each. These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs. Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret. Here we describe a different cysteinyl-tRNA synthetase from M. jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo. This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin. The existence of this protein contrasts with proposals that aminoacylation with cysteine in M. jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase.     

Coevolution of codon usage and transfer RNA abundance

The use of synonymous codons is strongly biased in the bacterium Escherichia coli and yeast, comprising both bias between codons recognized by the same transfer RNA and bias between groups of codons recognized by different synonymous tRNAs. A major determinant of the second sort of bias is tRNA content, codons recognized by abundant tRNAs being used more often than those recognised by rare tRNAs, particularly in highly expressed genes, probably owing to selection at the level of translation against codons recognized by rare tRNAs. Conversely, codon usage is likely to exert selection pressure on tRNA abundance. Here I develop a model for the coevolution of codon usage and tRNA abundance which explains why there are unequal abundances of synonymous tRNAs leading to biased usage between groups of codons recognized by them in unicellular organisms.     

Kinetic parameters of oscillating reaction of amino acid- BrO3^——Mn^2＋-H2SO4-acetone system

In the previous papers[1—3], the oscillating reactionusing amino acids as organic substrates was studied. Inorder to obtain information about the kinetic parameters ofoscillating reaction of amino acids in amino acid- BrO3 - ?Mn2 -H2SO4-acetone sy…     

Effect of alanine versus glycine in alpha-helices on protein stability.

The rational design of proteins requires knowledge of the helix-forming propensities (s-values) of the different amino acids. There is, however, considerable controversy about the relative values for alanine and glycine. We find from experiments on mutants of barnase that the relative effect of Ala versus Gly on helix stability depends crucially on the position in the helix (whether they are at the ends (caps) or are internal) and the context (the influence of their neighbours). Glycine is greatly preferred at the N and C caps. At internal positions, Ala stabilizes the helix relative to Gly by 0.4 to 2 kcal mol-1. The variation results from a combination of burial of hydrophobic surface on folding and interference with hydrogen bonding of the protein with solvent. There is a good empirical correlation between the relative stabilizing effects of Ala and Gly with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Gly to Ala. It is not valid to assign to each amino acid a unique s-value that is generally applicable to all positions in all helices in all proteins.