Kegg Pathway: Aminoacyl-tRNA biosynthesis

Biochemistry. 2008 Jul 22; 47(29): 7610-6
Cathopoulis TJ, Chuawong P, Hendrickson TL

Elongation factor Tu (EF-Tu) binds and loads elongating Aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNA (Gln) and Asn-tRNA (Asn) by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNA (Gln) and Asp-tRNA (Asn) as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNA (Asn) and predicted a similar discriminatory response against Glu-tRNA (Gln) [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194-6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420-3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNA (Gln) or Asp-tRNA (Asn), and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNA (Gln), like Asp-tRNA (Asn), will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these Aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.

J Mol Biol. 2008 Jul 11; 380(3): 451-64
Singh NS, Ahmad R, Sangeetha R, Varshney U

Translating ribosomes often stall during elongation. The stalled ribosomes are known to be recycled by tmRNA (SsrA)-mediated trans-translation. Another process that recycles the stalled ribosomes is characterized by peptidyl-tRNA release. However, the mechanism of peptidyl-tRNA release from the stalled ribosomes is not well understood. We used a defined system of an AGA-minigene containing a small open reading frame (ATG AGA AGA). Translation of the AGA-minigene mRNA is toxic to Escherichia coli because it stalls ribosomes during elongation and sequesters tRNA(Arg4) as a short-chain peptidyl-tRNA(Arg4) in the ribosomal P-site. We show that a ribosome recycling factor (RRF)-mediated process rescues the host from the AGA-minigene toxicity by releasing the peptidyl-tRNA(Arg4) from the ribosomes. The growth phenotypes of E. coli strains harboring mutant alleles of RRF and initiation factor 3 (IF3) genes and their consequences on lambdaimmP22 phage replication upon AGA-minigene expression reveal that IF3 facilitates the RRF-mediated processing of the stalled ribosomes. Additionally, we have designed a uracil DNA glycosylase gene construct, ung-stopless, whose expression is toxic to E. coli. We show that the RRF-mediated process also alleviates the ung-stopless construct-mediated toxicity to the host by releasing the ung mRNA from the ribosomes harboring long-chain peptidyl-tRNAs.

Mol Cell. 2008 Jun 6; 30(5): 589-98
Johansson M, Bouakaz E, Lovmar M, Ehrenberg M

The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate Aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of Aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.

RNA. 2008 Jul; 14(7): 1390-8
Goto Y, Murakami H, Suga H

Here we report experimental evidence that the translation initiation apparatus accepts D-amino acids ((D)aa), as opposed to only L-methionine, as initiators. Nineteen (D)aa, as the stereoisomers to their natural L-amino acids, were charged onto initiator tRNA(fMet)(CAU) using flexizyme technology and tested for initiation in a reconstituted Escherichia coli translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to (D)aa. Remarkably, all (D)aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with (D)aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding (D)aa-tRNA(fMet)(CAU). Although the inefficient formylation of (D)aa-tRNA(fMet)(CAU) resulted in modest expression of the corresponding peptide, preacetylation of (D)aa-tRNA(fMet)(CAU) dramatically increased expression level, implying that the formylation efficiency is one of the critical determinants of initiation efficiency with (D)aa. Our findings provide not only the experimental evidence that translation initiation tolerates (D)aa, but also a new means for the mRNA-directed synthesis of peptides capped with (D)aa or acyl-(D)aa at the N terminus.

J Mol Biol. 2008 Jun 27; 380(1): 193-205
Szaflarski W, Vesper O, Teraoka Y, Plitta B, Wilson DN, Nierhaus KH

We describe the optimization of a poly(Phe) synthesis system, the conditions of which have been applied for efficient translation of heteropolymeric mRNAs. Here we identify two parameters that are essential to obtain translation at efficiency and accuracy levels equivalent to those in vivo, viz., the fine-tuning of the energy-rich components with an acetyl-phosphate substrate for energy regeneration, as well as the ionic conditions. Applying this system revealed a number of new features: (i) 70S ribosomes are able to recycle within 300 s in a non-enzymatic fashion in the absence of tmRNA. This observation might explain the fact that a knockout of the tmRNA gene ssrA is not lethal for Escherichia coli cells in contrast to other bacterial strains, such as Bacillus subtilis. (ii) The high efficiency of the system was exploited to analyze the misincorporation of various amino acids (resolution limit=1:15,000). No misreading was observed at the middle codon position and only marginal effects were observed at the first one (even when misreading was artificially stimulated 20- to 30-fold), yielding an improved definition of the near-cognate and non-cognate Aminoacyl-tRNAs. (iii) Aminoglycosides increase Phe and Lys incorporation about 2-fold in the presence of poly(U) or poly(UUC) and poly(A), respectively, and induce a back-translocation (except hygromycin B) exclusively in the absence of EF-G*GTP, as do the non-related drugs viomycin and edeine.

Biochem Biophys Res Commun. 2008 Aug 1; 372(3): 480-5
Fukunaga R, Harada Y, Hirao I, Yokoyama S

An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K(m)=47.1muM and a k(cat)=0.151s(-1), which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA(Cys) with the GCA anticodon (26.9muM and 0.111s(-1)). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.

Proc Natl Acad Sci U S A. 2008 May 27; 105(21): 7428-33
Bullock TL, Rodríguez-Hernández A, Corigliano EM, Perona JJ

Information transfer from nucleic acid to protein is mediated by Aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K(M) of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA(Gln) by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA(Gln) by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA(Gln) complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.

Mol Cell Biol. 2008 Jul; 28(13): 4227-39
Doronina VA, Wu C, de Felipe P, Sachs MS, Ryan MD, Brown JD

"2A" oligopeptides are autonomous elements containing a D(V/I)EXNPGP motif at the C terminus. Protein synthesis from an open reading frame containing an internal 2A coding sequence yields two separate polypeptides, corresponding to sequences up to and including 2A and those downstream. We show that the 2A reaction occurs in the ribosomal peptidyltransferase center. Ribosomes pause at the end of the 2A coding sequence, over the glycine and proline codons, and the nascent chain up to and including this glycine is released. Translation-terminating release factors eRF1 and eRF3 play key roles in the reaction. On the depletion of eRF1, a greater proportion of ribosomes extend through the 2A coding sequence, yielding the full-length protein. In contrast, impaired eRF3 GTPase activity leads to many ribosomes failing to translate beyond 2A. Further, high-level expression of a 2A peptide-containing protein inhibits the growth of cells compromised for release factor activity and leads to errors in stop codon recognition. We propose that the nascent 2A peptide interacts with ribosomes to drive a highly unusual and specific "termination" reaction, despite the presence of a proline codon in the A site. After this, the majority of ribosomes continue translation, generating the separate downstream product.

Anticancer Drugs. 2008 Apr; 19(4): 391-9
Mönkkönen H, Kuokkanen J, Holen I, Evans A, Lefley DV, Jauhiainen M, Auriola S, Mönkkönen J

Bisphosphonates (BPs) are effective inhibitors of tumor-induced bone resorption. Recent studies have demonstrated that BPs inhibit growth, attachment and invasion of cancer cells in culture and promote apoptosis. The mechanisms responsible for the observed anti-tumor effects of BPs are beginning to be elucidated. Recently, we reported that nitrogen-containing bisphosphonates (N-BPs) induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of farnesyl diphosphate synthase in the mevalonate pathway. Similar to AppCp-type metabolites of non-N-BPs, ApppI is able to induce apoptosis. This study investigated BP-induced ATP analog formation and its effect on cancer cell growth. To evaluate zoledronic acid (a N-BP)-induced ApppI accumulation, inhibition of protein prenylation and clodronate (a non-N-BP) metabolism to AppCCl2p, MCF-7 and MDA-MB-436 breast cancer cells, MCF-10A nonmalignant breast cells, PC-3 prostate cancer cells, MG-63 osteosarcoma cells, RPMI-8226, and NCI-H929 myeloma cells were treated with 25 micromol/l zoledronic acid or 500 micromol/l clodronate for 24 h. The inhibition of cell growth by zoledronic acid and clodronate was studied in MCF-7, MDA-MB-436, and RPMI-8226 cells by exposing the cells with 1-100 micromol/l zoledronic acid or 10-2000 micromol/l clodronate for 72 h. Marked differences in zoledronic acid-induced ApppI formation and clodronate metabolism between the cancer cell lines were observed. The production of cytotoxic ATP analogs in tumor cells after BP treatment is likely to depend on the activity of enzymes, such as farnesyl diphosphate synthase or Aminoacyl-tRNA synthetases, responsible for ATP analog formation. Additionally, the potency of clodronate to inhibit cancer cell growth corresponds to ATP analog formation.

Nature. 2008 May 1; 453(7191): 120-3
Randau L, Schröder I, Söll D

The universality of ribonuclease P (RNase P), the ribonucleoprotein essential for transfer RNA (tRNA) 5' maturation, is challenged in the archaeon Nanoarchaeum equitans. Neither extensive computational analysis of the genome nor biochemical tests in cell extracts revealed the existence of this enzyme. Here we show that the conserved placement of its tRNA gene promoters allows the synthesis of leaderless tRNAs, whose presence was verified by the observation of 5' triphosphorylated mature tRNA species. Initiation of tRNA gene transcription requires a purine, which coincides with the finding that tRNAs with a cytosine in position 1 display unusually extended 5' termini with an extra purine residue. These tRNAs were shown to be substrates for their cognate Aminoacyl-tRNA synthetases. These findings demonstrate how nature can cope with the loss of the universal and supposedly ancient RNase P through genomic rearrangement at tRNA genes under the pressure of genome condensation.

Proc Natl Acad Sci U S A. 2008 May 6; 105(18): 6543-7
Garg RP, Qian XL, Alemany LB, Moran S, Parry RJ

The antibiotic valanimycin is a naturally occurring azoxy compound produced by Streptomyces viridifaciens MG456-hF10. Precursor incorporation experiments showed that valanimycin is derived from l-valine and l-serine via the intermediacy of isobutylamine and isobutylhydroxylamine. Enzymatic and genetic investigations led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes. A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene (vlmL) that encodes a class II seryl-tRNA synthetase. Previous studies suggested that the role of this enzyme is to provide seryl-tRNA for the valanimycin biosynthetic pathway. Here, we report the results of investigations to elucidate the role of seryl-tRNA in valanimycin biosynthesis. A combination of enzymatic and chemical studies has revealed that the VlmA protein encoded by the valanimycin biosynthetic gene cluster catalyzes the transfer of the seryl residue from seryl-tRNA to the hydroxyl group of isobutylhydroxylamine to produce the ester O-seryl-isobutylhydroxylamine. These findings provide an example of the involvement of an Aminoacyl-tRNA in an antibiotic biosynthetic pathway.

Biochem Biophys Res Commun. 2008 Jul 4; 371(3): 395-400
Lee YS, Han JM, Son SH, Choi JW, Jeon EJ, Bae SC, Park YI, Kim S

AIMP1 (also known as p43) is a factor associated with a macromolecular Aminoacyl-tRNA synthetase (ARS) complex but also plays diverse regulatory roles in various physiological processes. Here, we report that AIMP1 negatively regulates TGF-beta signaling via stabilization of Smurf2. TGF-beta-dependent phosphorylation and nuclear localization of R-Smads, induction of target genes, and growth arrest were increased in AIMP1-deficient or -suppressed cells. In AIMP1-deficient or suppressed cells, the Smurf2 level was decreased. Various binding assays demonstrated the direction interaction of the C-terminal region of AIMP1 directly with the Smad7-binding region of Smurf2. The association of Smurf2 with Smad7 and its ubiquitination were inhibited by AIMP1, thereby protecting its autocatalytic degradation stimulated by Smad7. Thus, this work suggests the novel activity of AIMP1 as a component of negative feedback loop of TGF-beta signaling.

Proc Natl Acad Sci U S A. 2008 Apr 29; 105(17): 6481-5
Pujol C, Bailly M, Kern D, Maréchal-Drouard L, Becker H, Duchêne AM

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by Aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA(Gln) is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA(Gln) is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA(Gln), which is the only tRNA(Gln) present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA(Gln) formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.

Mol Cell. 2008 Apr 25; 30(2): 190-202
Vazquez-Laslop N, Thum C, Mankin AS

Inducible expression of the erm erythromycin resistance genes relies on drug-dependent ribosome stalling. The molecular mechanisms underlying stalling are unknown. We used a cell-free translation system to elucidate the contribution of the nascent peptide, the drug, and the ribosome toward formation of the stalled complex during translation of the ermC leader cistron. Toe-printing mapping, selective amino acid labeling, and mutational analyses revealed the peptidyl transferase center (PTC) as the focal point of the stalling mechanism. In the ribosome exit tunnel, the C-terminal sequence of the nascent peptide, critical for stalling, is in the immediate vicinity of the universally conserved A2062 of 23S rRNA. Mutations of this nucleotide eliminate stalling. Because A2062 is located in the tunnel, it may trigger a conformational change in the PTC, responding to the presence of a specific nascent peptide. The cladinose-containing macrolide antibiotic in the tunnel positions the nascent peptide for interaction with the tunnel sensory elements.

Nat Struct Mol Biol. 2008 May; 15(5): 507-14
Zhang CM, Liu C, Slater S, Hou YM

Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNA(Cys) by an indirect pathway, whereby O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA(Cys) with phosphoserine (Sep), and Sep-tRNA-Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNA(Cys) tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1alpha or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNA(Cys).

Biochemistry. 2008 May 20; 47(20): 5638-48
Huang LY, Umanah G, Hauser M, Son C, Arshava B, Naider F, Becker JM

Ste2p is the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha factor of Saccharomyces cerevisiae. This receptor-pheromone pair has been used extensively as a paradigm for investigating GPCR structure and function. Expression in yeast harboring a cognate tRNA/Aminoacyl-tRNA synthetase pair specifically evolved to incorporate p-benzoyl- l-phenylalanine (Bpa) in response to the amber codon allowed the biosynthesis of Bpa-substituted Ste2p in its native cell. We replaced natural amino acid residues in Ste2p with Bpa by engineering amber TAG stop codons into STE2 encoded on a plasmid. Several of the expressed Bpa-substituted Ste2p receptors exhibited high-affinity ligand binding, and incorporation of Bpa into Ste2p influenced biological activity as measured by growth arrest of whole cells in response to alpha factor. We found that, at concentrations of 0.1-0.5 mM, a dipeptide containing Bpa could be used to enhance delivery of Bpa into the cell, while at 2 mM, both dipeptide and Bpa were equally effective. The application of a peptide delivery system for unnatural amino acids will extend the use of the unnatural amino acid replacement methodology to amino acids that are impermeable to yeast. Incorporation of Bpa into Ste2p was verified by mass spectrometric analysis, and two Bpa-Ste2p mutants were able to selectively capture alpha factor into the ligand-binding site after photoactivation. To our knowledge, this is the first experimental evidence documenting an unnatural amino acid replacement in a GPCR expressed in its native environment and the use of a mutated receptor to photocapture a peptide ligand.

In Silico Biol. 2007; 7(3): 309-18
Rodin AS, Rodin SN

The updated structural and phylogenetic analyses of tRNA pairs with complementary anticodons provide independent support for our earlier finding, namely that these tRNA pairs concertedly show complementary second bases in the acceptor stem. Two implications immediately follow: first, that a tRNA molecule gained its present, complete, cloverleaf shape via duplication(s) of a shorter precursor. Second, that common ancestry is shared by two major components of the genetic code within the tRNA molecule--the classic code per se embodied in anticodon triplets, and the operational code of aminoacylation embodied primarily in the first three base pairs of the acceptor stems. In this communication we show that it might have been a double, sense-antisense, in-frame translation of the very first protein-encoding genes that directed the code's earliest expansion, thus preserving this fundamental dual-complementary link between acceptors and anticodons. Furthermore, the dual complementarity appears to be consistent with two mirror-symmetrical modes by which class I and II Aminoacyl-tRNA synthetases recognize the cognate tRNAs--from the minor and major groove side of the acceptor stem, respectively.

Anal Biochem. 2008 Jul 1; 378(1): 90-2
Kugimiya A, Morii M, Ohtsuki T

The detection of amino acids using Aminoacyl-tRNA synthetases (ARSs) as the molecular recognition element was proposed, and the binding activity and specificity of ARSs were evaluated. Using this rapid and easy method, from 15 to 50 microM tyrosine could be measured specifically. The method suggested in this article could be realized without an amino acid labeling process or a large volume of organic solvents, and the time for measurement was reasonable.

Mol Cell. 2008 Apr 11; 30(1): 3-4
Ribas de Pouplana L, Geslain R

In a recent issue of Molecular Cell, Jia et al. (2008) demonstrate that time-controlled repression of interferon-induced transcripts depends upon the interaction between an RNA structure in these transcripts and protein domains harbored by a mammalian Aminoacyl-tRNA synthetase.

Biochemistry. 2008 Apr 29; 47(17): 4898-906
Trobro S, Aqvist J

The current view of ribosomal peptidyl transfer is that the ribosome is a ribozyme and that ribosomal proteins are not involved in catalysis of the chemical reaction. This view is largely based on the first crystal structures of bacterial large ribosomal subunits that did not show any protein components near the peptidyl transferase center (PTC). Recent crystallographic data on the full 70S ribosome from Thermus thermophilus, however, show that ribosomal protein L27 extends with its N-terminus into the PTC in accordance with independent biochemical data, thus raising the question of whether the ribozyme picture is strictly valid. We have carried out extensive computer simulations of the peptidyl transfer reaction in the T. thermophilus ribosome to address the role of L27. The results show a reaction rate similar to that obtained in earlier simulations of the Haloarcula marismortui reaction. Furthermore, deletion of L27 is predicted to only give a minor rate reduction, in agreement with biochemical data, suggesting that the ribozyme view is indeed valid. The N-terminus of L27 is predicted to interact with the A76 phosphate group of the A-site tRNA, thereby explaining the observed impairment of A-site substrate binding for ribosomes lacking L27. Simulations are also reported for the reaction with puromycin, an A-site tRNA analogue which lacks the A76 phosphate group. The calculated energetics shows that this substrate can cause a downward p K a shift of L27 and that the reaction proceeds faster with the L27 N-terminus deprotonated, in contrast to the situation with Aminoacyl-tRNA substrates. These results could explain the observed differences in pH dependence between the puromycin and C-puromycin reactions, where the former reaction has been seen to depend on an additional ionizing group besides the attacking amine, and our model predicts this ionizing group to be the N-terminal amine of L27.