A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding.

Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20-70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNA(Asp), we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identifies a lysine-rich 11 residue sequence ((29)LSKKALKKLQK(39) in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide.

tRNA mimics.

Mimics recapitulating the structural features of tRNAs are involved in biological processes other than ribosome-dependent protein synthesis. A knowledge of the rules underlying the architecture and function of tRNAs allows the design of non-natural mimics. The study of these mimics sheds light upon links between replication, translation and metabolic pathways, leads to biotechnological applications, and provides experimental and conceptual tools for the exploration of primordial evolutionary processes.

Sequences outside recognition sets are not neutral for tRNA aminoacylation. Evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe.

Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363-1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia coli tRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a "classical" recognition element since its antideterminant effect is compensated for by a second-site mutation. This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name "permissive elements," are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently.

Subtle atomic group discrimination in the RNA minor groove.

As a problem in molecular recognition and for drug discovery, great interest has developed around the possibility that RNA structures could be discriminated by peptides and other small molecules. Although small peptides have been shown to have the capacity to discriminate specific bulges and loops in RNA molecules, discrimination of double helical regions by a peptide binder has not been reported. Indeed, the most accessible part of an RNA helix is the minor groove, and fundamental stereochemical considerations have suggested that discrimination of at least some base pairs would be difficult in the minor groove. Here we report the design and isolation of a peptide binder that manifests the most subtle kind of discrimination of base pair differences in the RNA minor groove. Functional discrimination of a single atomic group is demonstrated as well as the difference between two different angular orientations of the same group. This report of RNA helix discrimination by a peptide binder suggests a richer potential for RNA minor groove recognition than previously thought.

Peptides for RNA discrimination and for assembly of enzymes that act on RNA.

RNA recognition by tRNA synthetases is thought to have arisen by the recruitment of RNA binding peptide elements into the frameworks of primordial enzymes that carried out amino acid activation. While peptides have been shown to have the capacity to discriminate irregular RNA structures such as bulges and loops, the sequence-specific recognition of base pairs in RNA helices like those in tRNAs had not been demonstrated. Such discrimination by peptide binders was thought to be inherently difficult. But in this work we show that discrimination of a single base pair in an RNA helix may be achieved with a rationally designed, chemically synthesized peptide. In a separate study, we demonstrated the noncovalent association of a peptide binder with an amino acid activation domain to give an active and specific tRNA synthetase. Thus, peptides with high specificity for an RNA helix can be obtained. In addition, a peptide can associate with another protein to give an enzyme that acts on RNA. The principles suggested by these studies may be general.

Evidence that two present-day components needed for the genetic code appeared after nucleated cells separated from eubacteria.

The trinucleotide/amino acid relationships of the present-day genetic code are established by the amino-acylation reactions of tRNA synthetases, whereby each of 20 specific amino acids is attached to its cognate tRNAs, which bear anticodon trinucleotides. Because of its universality, the appearance of the modern genetic code is thought to predate the separation of prokaryotic and eukaryotic organisms in the universal phylogenetic tree. In the light of new sequence information, we present here a phylogenetic analysis that shows an unusual picture for tyrosyl- and tryptophanyl-tRNA synthetases. Ij particular, the eukaryotic tyrosyl- and tryptophanyl-tRNA synthetases are more related to each other than to their respective prokaryotic counterparts. In contrast, each of the other 18 eukaryotic synthetases is more related to its prokaryotic counterpart than to any eukaryotic synthetase specific for a different amino acid. Our results raise the possibility that present day tyrosyl- and tryptophanyl-tRNA synthetases appeared after the separation of nucleated cells from eubacteria. The results have implications for the development of the genetic code.

Identity switches between tRNAs aminoacylated by class I glutaminyl- and class II aspartyl-tRNA synthetases.

High-resolution X-ray structures for the tRNA/aminoacyl-tRNA synthetase complexes between Escherichia coli tRNAGln/GlnRS and yeast tRNAAsp/AspRS have been determined. Positive identity nucleotides that direct aminoacylation specificity have been defined in both cases; E. coli tRNAGln identity is governed by 10 elements scattered in the tRNA structure, while specific aminoacylation of yeast tRNAAsp is dependent on 5 positions. Both identity sets are partially overlapping and share 3 nucleotides. Interestingly, the two enzymes belong to two different classes described for aminoacyl-tRNA synthetases. The class I glutaminyl-tRNA synthetase and the class II aspartyl-tRNA synthetase recognize their cognate tRNA from opposite sides. Mutants derived from glutamine and aspartate tRNAs have been created by progressively introducing identity elements from one tRNA into the other one. Glutaminylation and aspartylation assays of the transplanted tRNAs show that identity nucleotides from a tRNA originally aminoacylated by a synthetase from one class are still recognized if they are presented to the enzyme in a structural framework corresponding to a tRNA aminoacylated by a synthetase belonging to the other class. The simple transplantation of the glutamine identity set into tRNAAsp is sufficient to obtain glutaminylatable tRNA, but additional subtle features seem to be important for the complete conversion of tRNAGln in an aspartylatable substrate. This study defines C38 in yeast tRNAAsp as a new identity nucleotide for aspartylation. We show also in this paper that, during the complex formation, aminoacyl-tRNA synthetases are at least partially responsible for conformational changes which involve structural constraints in tRNA molecules.

Synthetic polyamines stimulate in vitro transcription by T7 RNA polymerase.

The influence of nine synthetic polyamines on in vitro transcription with T7 RNA polymerase has been studied. The compounds used were linear or macrocyclic tetra- and hexaamine, varying in their size, shape and number of protonated groups. Their effect was tested on different types of templates, all presenting the T7 RNA promoter in a double-stranded form followed by sequences encoding short transcripts (25 to 35-mers) either on single- or double-stranded synthetic oligodeoxyribonucleotides. All polyamines used stimulate transcription of both types of templates at levels dependent on their size, shape, protonation degree, and concentration. For each compound, an optimal concentration could be defined; above this concentration, transcription inhibition occurred. Highest stimulation (up to 12-fold) was obtained by the largest cyclic compound called [38]N6C10.

Efficient aminoacylation of resected RNA helices by class II aspartyl-tRNA synthetase dependent on a single nucleotide.

We show here that small RNA helices which recapitulate part or all of the acceptor stem of yeast aspartate tRNA are efficiently aminoacylated by cognate class II aspartyl-tRNA synthetase. Aminoacylation is strongly dependent on the presence of the single-stranded G73 'discriminator' identity nucleotide and is essentially insensitive to the sequence of the helical region. Substrates which contain as few as 3 bp fused to G73CCAOH are aspartylated. Their charging is insensitive to the sequence of the loop closing the short helical domains. Aminoacylation of the aspartate mini-helix is not stimulated by a hairpin helix mimicking the anticodon domain and containing the three major anticodon identity nucleotides. A thermodynamic analysis demonstrates that enzyme interactions with G73 in the resected RNA substrates and in the whole tRNA are the same. Thus, if the resected RNA molecules resemble in some way the earliest substrates for aminoacylation with aspartate, then the contemporary tRNA(Asp) has quantitatively retained the influence of the major signal for aminoacylation in these substrates.

Triple aminoacylation specificity of a chimerized transfer RNA.

We report here the rational design and construction of a chimerized transfer RNA with tripartite aminoacylation specificity. A yeast aspartic acid specific tRNA was transformed into a highly efficient acceptor of alanine and phenylalanine and a moderate acceptor of valine. The transformation was guided by available knowledge of the requirements for aminoacylation by each of the three amino acids and was achieved by iterative changes in the local sequence context and the structural framework of the variable loop and the two variable regions of the dihydrouridine loop. The changes introduced to confer efficient acceptance of the three amino acids eliminate aminoacylation with aspartate. The interplay of determinants and antideterminants for different specific aminoacylations, and the constraints imposed by the structural framework, suggest that a tRNA with an appreciable capacity for more than three efficient aminoacylations may be inherently difficult to achieve.

Anticodon-independent aminoacylation of an RNA minihelix with valine.

Minihelices mimicking the amino acid acceptor and anticodon branches of yeast tRNA(Val) have been synthesized by in vitro transcription of synthetic templates. It is shown that a minihelix corresponding to the amino acid acceptor branch and containing solely a valine-specific identity nucleotide can be aminoacylated by yeast valyl-tRNA synthetase. Its charging ability is lost after mutating this nucleotide. This ability is stimulated somewhat by the addition of a second hairpin helix that mimicks the anticodon arm, which suggests that information originating from the anticodon stem-loop can be transmitted to the active site of the enzyme by the core of the protein.