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P Schimmel

Publications and source records attributed to P Schimmel.

At least 19 recordsLinked to original sources
PubMed2000

One of two genes encoding glycyl-tRNA synthetase in Saccharomyces cerevisiae provides mitochondrial and cytoplasmic functions.

In the yeast Saccharomyces cerevisiae, two genes (GRS1 and GRS2) encode glycyl-tRNA synthetase (GlyRS1 and GlyRS2, respectively). 59% of the sequence of GlyRS2 is identical to that of GlyRS1. Others have proposed that GRS1 and GRS2 encode the cytoplasmic and mitochondrial enzymes, respectively. In this work, we show that GRS1 encodes both functions, whereas GRS2 is dispensable. In addition, both cytoplasmic and mitochondrial phenotypes of the knockout allele of GRS1 in S. cerevisiae are complemented by the expression of the only known gene for glycyl-tRNA synthetase in Schizosaccharomyces pombe. Thus, a single gene for glycyl-tRNA synthetase likely encodes both cytoplasmic and mitochondrial activities in most or all yeast. Phylogenetic analysis shows that GlyRS2 is a predecessor of all yeast GlyRS homologues. Thus, GRS1 appears to be the result of a duplication of GRS2, which itself is pseudogene-like.

Amino Acid Sequence↗
PubMed2000

Errors from selective disruption of the editing center in a tRNA synthetase.

Some aminoacyl-tRNA synthetases have two catalytic centers that together achieve fine-structure discrimination of closely similar amino acids. The role of tRNA is to stimulate translocation of a misactivated amino acid from the active site to the editing site where the misactivated substrate is eliminated by hydrolysis. Using isoleucyl-tRNA synthetase as an example, we placed mutations in the catalytic center for editing at residues strongly conserved from bacteria to humans. A particular single substitution and one double substitution resulted in production of mischarged tRNA, by interfering specifically with the chemical step of hydrolytic editing. The substitutions affected neither amino acid activation nor aminoacylation, with the cognate amino acid. Thus, because of the demonstrated functional independence of the two catalytic sites, errors of aminoacylation can be generated by selective mutations in the center for editing.

Amino Acid Sequence↗
PubMed2000

Functional redundancy in the nonspecific RNA binding domain of a class I tRNA synthetase.

The sequence of a 228-amino acid nonspecific RNA binding domain appended to the N terminus of a eukaryote tRNA synthetase is shown here to have two lysine-rich clusters (LRCs) that are functionally significant in vivo and in vitro. These two LRCs have unrelated sequences and are separated by a spacer of over 100 amino acids. By using a sensitive test for function in vivo, each LRC is shown to be sufficient in the absence of the other. This sufficiency requires fusion of the spacer to either of the LRCs. Experiments in vitro confirmed that the LRCs are each important for RNA binding. Thus, this nonspecific RNA binding domain has two dissimilar lysine-rich sequence elements that are functionally redundant. Further experiments suggest that this redundancy is not used to dock two molecules of RNA but rather to enhance the overall affinity for a single RNA molecule.

Amino Acyl-tRNA Synthetases↗
PubMed2000

Exiting an RNA world.

The RNA world hypothesis gains support from the in vitro evolution of a bifunctional ribozyme that can recognize an activated glutaminyl ester and subsequently aminoacylate a tRNA molecule.

Acylation↗
PubMed2000

Functional defects of pathogenic human mitochondrial tRNAs related to structural fragility.

Aminoacylation of transfer RNAs (tRNAs) is essential for protein synthesis. A growing number of human diseases correlate with point mutations in tRNA genes within the mitochondrial genome. These tRNAs have unique sequences that suggest they have fragile structures. However, the structural significance of pathology-related tRNA mutations and their effects on molecular function have not been explored. Here, opthalmoplegia related mutants of a human mitochondrial tRNA have been investigated. Each mutation replaces either an A-U or G-C pair in the predicted secondary structure with an A-C pair. Aminoacylation of each mutant tRNA was severely attenuated. Moreover, each strongly inhibited aminoacylation of the wild type substrate, suggesting that the effects of these mutations might not be bypassed in the potentially heteroplasmic environment of mitochondria. The function of mutant tRNAs was rescued by single compensatory mutations that restored Watson-Crick base pairing and reintroduced stability into regions of predicted secondary structure, even though the pairs introduced were different from those found in the wild type tRNA. Thus, functional defects caused by a subset of pathogenic mutations may result from the inherent structural fragility of human mitochondrial tRNAs.

Humans↗
PubMed1999

Nucleotide determinants for tRNA-dependent amino acid discrimination by a class I tRNA synthetase.

The high accuracy of the genetic code relies on the ability of tRNA synthetases to discriminate rigorously between closely similar amino acids. While the enzymes can detect differences between closely similar amino acids at an accuracy of about 1 part in 100-200, a finer discrimination requires the presence of the cognate tRNA. The role of the tRNA is to direct the misactivated amino acid to a distinct catalytic site for editing where hydrolysis occurs. Previous work showed that three nucleotides at the corner of the L-shaped tRNA were collectively required. Here we show that each of these nucleotides individually contributes to the efficiency of editing. However, all are dispensable for the chemical step of hydrolysis. Instead, these nucleotides are required for translocation of a misactivated amino acid from the active site to the center for editing.

Acylation↗
PubMed1999

Major anticodon-binding region missing from an archaebacterial tRNA synthetase.

The small size of the archaebacterial Methanococcus jannaschii tyrosyl-tRNA synthetase may give insights into the historical development of tRNAs and tRNA synthetases. The L-shaped tRNA has two major arms-the acceptor.TpsiC minihelix with the amino acid attachment site and the anticodon-containing arm. The structural organization of the tRNA synthetases parallels that of tRNAs. The more ancient synthetase domain contains the active site and insertions that interact with the minihelix portion of the tRNA. A second, presumably more recent, domain interacts with the anticodon-containing section of tRNA. The small size of the M. jannaschii enzyme is due to the absence of most of the second domain, including a segment thought to bind to the anticodon. Consistent with the absence of an anticodon-binding motif, a mutation of the central base of the anticodon had a relatively small effect on the aminoacylation efficiency of the M. jannaschii enzyme. In contrast, others showed earlier that the same mutation severely reduced charging by a normal-sized bacterial enzyme that has the aforementioned anticodon-binding motif. However, the M. jannaschii enzyme has a peptide insertion into its catalytic domain. This insertion is shared with all other tyrosyl-tRNA synthetases and is needed for a critical minihelix interaction. We show that the M. jannaschii enzyme is active on minihelix substrates over a wide temperature range and has preserved the same peptide-dependent minihelix specificity seen in other tyrosine enzymes. These findings are consistent with the concept that anticodon interactions of tRNA synthetases were later adaptations to the emerging synthetase-tRNA complex that was originally framed around the minihelix.

Anticodon↗
PubMed1999

Evidence for breaking domain-domain functional communication in a synthetase-tRNA complex.

We report here evidence for mutations that break domain-domain functional communication in a synthetase-tRNA complex. Each synthetase is roughly divided into two major domains that are paralleled by the two arms of the L-shaped tRNA structure. The active-site-containing domain interacts with the acceptor arm of the tRNA. The second domain frequently interacts with the anticodon-containing arm. By an induced-fit mechanism, contacts with the anticodon can activate formation of a robust transition state at a site over 70 A away. This induced-fit-based activation is thought to occur through domain-domain signaling and is seen by the enhancement of aminoacylation of the anticodon-containing full tRNA versus a substrate based on the acceptor arm alone. Here we describe a rationally designed mutant methionyl-tRNA synthetase containing two point substitutions at sites that potentially link an anticodon-binding motif to the catalytic domain. The double mutation had no effect on interactions with either the isolated acceptor arm or the anticodon stem-loop. In contrast to interactions with the separate pieces, the mutant enzyme was severely impaired for binding the native tRNA and lost much of its ability to enhance the rate of charging of the full tRNA over that of a substrate based on the acceptor arm alone. We propose that these residues are part of a network for facilitating domain-domain communication for formation of an active synthetase-tRNA complex by induced fit.

Acylation↗
PubMed1999

Domain-domain communication in a miniature archaebacterial tRNA synthetase.

The three-dimensional structure of tRNA is organized into two domains-the acceptor-TPsiC minihelix with the amino acid attachment site and a second, anticodon-containing, stem-loop domain. Aminoacyl-tRNA synthetases have a structural organization that roughly recapitulates the two-domain organization of tRNAs-an older primary domain that contains the catalytic center and interacts with the minihelix and a secondary, more recent, domain that makes contacts with the anticodon-containing arm. The latter contacts typically are essential for enhancement of the catalytic constant k(cat) through domain-domain communication. Methanococcus jannaschii tyrosyl-tRNA synthetase is a miniature synthetase with a tiny secondary domain suggestive of an early synthetase evolving from a one-domain to a two-domain structure. Here we demonstrate functional interactions with the anticodon-containing arm of tRNA that involve the miniaturized secondary domain. These interactions appear not to include direct contacts with the anticodon triplet but nonetheless lead to domain-domain communication. Thus, interdomain communication may have been established early in the evolution from one-domain to two-domain structures.

Acylation↗
PubMed1999

Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains.

An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of Escherichia coli alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1-2 kcal.mol(-1) of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.

Acylation↗
PubMed1999

Efficient 50S ribosome-catalyzed peptide bond synthesis with an aminoacyl minihelix.

RNA minihelices that recreate the amino acid acceptor domain of the two-domain L-shaped tRNA molecule are substrates for specific aminoacylation by tRNA synthetases. Some lines of evidence suggest that this domain arose independently of and predated the second, anticodon-containing domain. With puromycin and a minihelix charged with alanine, we show here efficient 50S ribosome catalyzed peptide synthesis. The aminoacyl minihelix is as active as aminoacyl tRNA in the synthetic reaction. The high efficiency of the charged minihelix is due to a relatively strong interaction with the 50S particle. In contrast, an aminoacyl RNA fragment that recreates the 3'-side of the tRNA acceptor stem has a much weaker interaction with the 50S particle. These results are consistent with the minihelix domain being the major loci for tRNA interactions with the 50S ribosome. They may also have implications for the historical development of RNA-based systems of peptide synthesis.

Acylation↗
PubMed1999

Highly differentiated motifs responsible for two cytokine activities of a split human tRNA synthetase.

While native human tyrosyl-tRNA synthetase (TyrRS) is inactive as a cell-signaling molecule, it can be split into two distinct cytokines. The enzyme is secreted under apoptotic conditions in culture where it is cleaved into an N-terminal fragment that harbors the catalytic site and into a C-domain fragment found only in the mammalian enzymes. The N-terminal fragment is an interleukin-8 (IL-8)-like cytokine, whereas the released C-domain is an endothelial-monocyte-activating polypeptide II (EMAP II)-like cytokine. Although the IL-8-like activity of the N-fragment depends on an ELR motif found in alpha-chemokines and conserved among mammalian TyrRSs, here we show that a similar (NYR) motif in the context of a lower eukaryote TyrRS does not confer the IL8-like activity. We also show that a heptapeptide from the C-domain has EMAP II-like chemotaxis activity for mononuclear phagocytes and polymorphonuclear leukocytes. Eukaryote proteins other than human TyrRS that have EMAP II-like domains have variants of the heptapeptide motif. Peptides based on these sequences are inactive as cytokines. Thus, the cytokine activities of split human TyrRS depend on highly differentiated motifs that are idiosyncratic to the mammalian system.

Amino Acid Sequence↗
PubMed1999

Structure-specific tRNA-binding protein from the extreme thermophile Aquifex aeolicus.

The genome of the bacterium Aquifex aeolicus encodes a polypeptide which is related to a small portion of a sequence found in one prokaryotic and two eukaryotic tRNA synthetases. It also is related to a portion of Arc1p, a tRNA-binding protein believed to be important for nuclear trafficking of tRNAs. Here we cloned, expressed and purified the 111 amino acid polypeptide (designated Trbp111) and showed by ultracentrifugation analysis that it is a stable dimer in solution. The protein was also crystallized in a monoclinic lattice. X-ray diffraction analysis at 2.8 A resolution revealed a prominent non-crystallographic 2-fold axis, consistent with the presence of a symmetric homodimeric structure. Band-shift analysis with polyacrylamide gels showed that the dimer binds tRNAs, but not RNA duplexes, RNA hairpins, single-stranded RNA nor 5S rRNA. Complex formation with respect to tRNA is non-specific, with a single tRNA bound per dimer. Thus, Trbp111 is a structure-specific tRNA-binding protein. These results and other considerations raise the possibility that Trbp111 is a tRNA-specific chaperone which stabilizes the native L-shaped fold in the extreme thermophile and which has been incorporated into much larger tRNA-binding proteins of higher organisms.

Amino Acid Sequence↗
PubMed1999

Species barrier to RNA recognition overcome with nonspecific RNA binding domains.

We show here that nonspecific RNA-protein interactions can significantly enhance the biological activity of an essential RNA. protein complex. Bacterial glutaminyl-tRNA synthetase poorly aminoacylates yeast tRNA and, as a consequence, cannot rescue a knockout allele of the gene for the yeast homologue. In contrast to the bacterial protein, the yeast enzyme has an extra appended domain at the N terminus. Previously, we showed that fusion of this yeast-specific domain to the bacterial protein enabled it to function as a yeast enzyme in vivo and in vitro. We suggested that the novel yeast-specific domain contributed to RNA interactions in a way that compensated for the poor fit between the yeast tRNA and bacterial enzyme. Here we establish that the novel appended domain by itself binds nonspecifically to different RNA structures. In addition, we show that fusion of an unrelated yeast protein, Arc1p, to the bacterial enzyme also converts it into a functional yeast enzyme in vivo and in vitro. A small C-terminal segment of Arc1p is necessary and sufficient for this conversion. This segment was shown by others to have nonspecific tRNA binding properties. Thus, nonspecific RNA binding interactions in general can compensate for barriers to formation of a specific and essential RNA.protein complex.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Different adaptations of the same peptide motif for tRNA functional contacts by closely homologous tRNA synthetases.

The N73 nucleotide at the end of the tRNA acceptor stem is commonly used by tRNA synthetases for discrimination. Because only a few synthetase-tRNA cocrystal structures have been determined, understanding of the molecular basis for N73 discrimination is limited. Here we investigated the possibility that, for at least some synthetases, the capacity to recognize different N73 nucleotides resides in the variable sequence of the loop of motif 2, a motif found in all class II enzymes. In the cocrystal of the class II yeast aspartyl-tRNA synthetase, atomic groups of the G73 discriminator of tRNAAsp interact with three side chains of the enzyme. We examined lysyl-tRNA synthetase, a close structural homologue of the aspartyl enzyme. Different substitutions were introduced into the Escherichia coli enzyme (A73 discriminator) to make its loop more like that of the human enzyme (G73 discriminator). Our data show that the loop of motif 2 of the lysine enzyme makes tRNA functional contacts, as predicted from the structural comparison. And yet, the E. coli enzyme with the "humanized" loop sequence had the same quantitative kinetic preference for A73 versus G as the wild-type enzyme. We conclude that discriminator base selectivity in the lysine enzyme requires residues in addition to or other than those in the loop of motif 2. Thus, even tRNA synthetases that are close structural homologues may use the same RNA binding element to make functional contacts with places (in the acceptor stem) that are idiosyncratic to each synthetase-tRNA pair.

Acylation↗