Charged tRNA Research Articles

High levels of tRNA abundance and alteration of tRNA charging by bortezomib in multiple myeloma

In multiple myeloma (MM), malignant plasma cells produce large amounts of antibodies and have highly active protein translational machinery. It is not known whether regulation of the abundance and aminoacylation (charging) of transfer RNA (tRNA) takes place in myeloma cells to accommodate for the increased amount of protein translation. Using tRNA-specific microarrays, we demonstrate that tRNA levels are significantly elevated in MM cell lines compared to normal bone marrow cells. We furthermore show that the addition of the proteasome inhibitor, bortezomib (Velcade™, PS-341) results in decreased charging levels of tRNAs, in particular those coding for hydrophobic amino acids. These results suggest that tRNA properties are altered in MM to accommodate for its increased need for protein translation, and that proteasome inhibition directly impacts protein synthesis in MM through effects on tRNA charging.

Defects in Transient tRNA Translocation Bypass tRNA Synthetase Quality Control Mechanisms

Quality control mechanisms during protein synthesis are essential to fidelity and cell survival. Leucyl-tRNA synthetase (LeuRS) misactivates non-leucine amino acids including isoleucine, methionine, and norvaline. To prevent translational errors, mischarged tRNA products are translocated 30A from the canonical aminoacylation core to a hydrolytic editing-active site within a completely separate domain. Because it is transient, the tRNA translocation mechanism has been difficult to isolate. We have identified a "translocation peptide" within Escherichia coli LeuRS. Mutations in the translocation peptide cause tRNA to selectively bypass the editing-active site, resulting in mischarging that is lethal to the cell. This bypass mechanism also rescues aminoacylation of an editing site mutation that hydrolyzes correctly charged Leu-tRNA(Leu). Thus, these LeuRS mutants charge tRNA(Leu) but fail to translocate these products to the hydrolytic site, where they are cleared to guard against genetic code ambiguities.

Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop

We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3'-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.

Quality control by the ribosome following peptide bond formation

The overall fidelity of protein synthesis has been thought to rely on the combined accuracy of two basic processes: the aminoacylation of transfer RNAs with their cognate amino acid by the aminoacyl-tRNA synthetases, and the selection of cognate aminoacyl-tRNAs by the ribosome in cooperation with the GTPase elongation factor EF-Tu. These two processes, which together ensure the specific acceptance of a correctly charged cognate tRNA into the aminoacyl (A) site, operate before peptide bond formation. Here we report the identification of an additional mechanism that contributes to high fidelity protein synthesis after peptidyl transfer, using a well-defined in vitro bacterial translation system. In this retrospective quality control step, the incorporation of an amino acid from a non-cognate tRNA into the growing polypeptide chain leads to a general loss of specificity in the A site of the ribosome, and thus to a propagation of errors that results in abortive termination of protein synthesis.

Crystal Structure of the Intact Archaeal Translation Initiation Factor 2 Demonstrates Very High Conformational Flexibility in the α- and β-Subunits

In Eukarya and Archaea, translation initiation factor 2 (eIF2/aIF2), which contains three subunits (α, β, and γ), is pivotal for binding of charged initiator tRNA to the small ribosomal subunit. The crystal structure of the full-sized heterotrimeric aIF2 from Sulfolobus solfataricus in the nucleotide-free form has been determined at 2.8-Å resolution. Superposition of four molecules in the asymmetric unit of the crystal and the comparison of the obtained structures with the known structures of the aIF2αγ and aIF2βγ heterodimers revealed high conformational flexibility in the α- and β-subunits. In fact, the full-sized aIF2 consists of a rigid central part, formed by the γ-subunit, domain 3 of the α-subunit, and the N-terminal α-helix of the β-subunit, and two mobile “wings,” formed by domains 1 and 2 of the α-subunit, the central part, and the zinc-binding domain of the β-subunit. High structural flexibility of the wings is probably required for interaction of aIF2 with the small ribosomal subunit. Comparative analysis of all known structures of the γ-subunit alone and within the heterodimers and heterotrimers in nucleotide-bound and nucleotide-free states shows that the conformations of switch 1 and switch 2 do not correlate with the assembly or nucleotide states of the protein.

Perturbation of the tRNA Tertiary Core Differentially Affects Specific Steps of the Elongation Cycle

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA(Phe), 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA.EF-Tu.GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.

Global Responses of Methanococcus maripaludis to Specific Nutrient Limitations and Growth Rate

Continuous culture, transcriptome arrays, and measurements of cellular amino acid pools and tRNA charging levels were used to determine the response of Methanococcus maripaludis to leucine limitation. For comparison, the responses to phosphate and H2 limitations were measured as well. In addition, the effect of growth rate was determined. Leucine limitation resulted in a broad response. tRNA(Leu) charging decreased, but only small increases in mRNA were seen for amino acid biosynthesis genes. However, the cellular levels of free isoleucine and valine showed significant increases, indicating a coordinate regulation of branched-chain amino acids at a post-mRNA level. Leucine limitation also resulted in increased mRNA abundance for ribosomal protein genes, increased rRNA abundance, and decreased mRNA abundance for genes of methanogenesis. In contrast, phosphate limitation induced a specific response, a marked increase in mRNA levels for a phosphate transporter. Some mRNA levels responded to more than one factor; for example, transcripts for flagellum synthesis genes decreased under conditions of leucine limitation and increased under H2 limitation. Increased growth rate resulted in increased mRNA levels for ribosomal protein genes, increased rRNA abundance, and increased mRNA for a gene encoding an S-layer protein.

An in silico analysis of T-box regulated genes and T-box evolution in prokaryotes, with emphasis on prediction of substrate specificity of transporters

BackgroundT-box anti-termination is an elegant and sensitive mechanism by which many bacteria maintain constant levels of amino acid-charged tRNAs. The amino acid specificity of the regulatory element is related to a so-called specifier codon and can in principle be used to guide the functional annotation of the genes controlled via the T-box anti-termination mechanism.ResultsHidden Markov Models were defined to search the T-box regulatory element and were applied to all completed prokaryotic genomes. The vast majority of the genes found downstream of the retrieved elements encoded functionalities related to transport and synthesis of amino acids and the charging of tRNA. This is completely in line with findings reported in literature and with the proposed biological role of the regulatory element. For several species, the functional annotation of a large number of genes encoding proteins involved in amino acid transport could be improved significantly on basis of the amino acid specificity of the identified T-boxes. In addition, these annotations could be extrapolated to a larger number of orthologous systems in other species. Analysis of T-box distribution confirmed that the element is restricted predominantly to species of the phylum Firmicutes. Furthermore, it appeared that the distribution was highly species specific and that in the case of amino acid transport some boxes seemed to "pop-up" only recently.ConclusionWe have demonstrated that the identification of the molecular specificity of a regulatory element can be of great help in solving notoriously difficult annotation issues, e.g. by defining the substrate specificity of genes encoding amino acid transporters on basis of the amino acid specificity of the regulatory T-box. Furthermore, our analysis of the species-dependency of the occurrence of specific T-boxes indicated that these regulatory elements propagate in a semi-independent way from the genes that they control.

Total Aminoacyl-Transfer RNA Pool Is Greater in Liver Than Muscle in Rabbits

Transfer RNA (tRNA)-charged amino acids are direct precursors of protein synthesis. Therefore, the amount and profile of amino acids in the aminoacyl-tRNA pool may be closely related to the rate of protein synthesis in the tissue. This study was designed to compare the aminoacyl-tRNA pools in liver and muscle, 2 distinct tissues with different rates of protein synthesis. Liver and muscle samples were taken from 6 rabbits and aminoacyl-tRNA was isolated with sequential acid-phenol:chloroform extraction, followed by total RNA and tRNA purification. Amino acids in the aminoacyl-tRNA pool were measured by HPLC after deacylation. Liver contained 3.4 times more tRNA than muscle (585 ± 120 vs. 132 ± 11 μg of tRNA/g of tissue; P < 0.001). Overall tRNA charging was also greater in liver (14.22 ± 4.42 nmol of amino acids/mg of tRNA) than in muscle (7.00 ± 1.76 nmol of amino acids/mg of tRNA) (P < 0.05). The greater availability and charging efficiency of tRNA in liver as compared with muscle may influence the extent to which amino acid precursor availability regulates protein synthesis in these 2 tissues.

Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial alpha-subunit of PheRS and the B8 domain of its beta-subunit. Together, the alpha-subunit and the 'RNP-domain' (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 55, b = 90, c = 96 A.

New Insights into the Interactions of the Translation Initiation Factor 2 from Archaea with Guanine Nucleotides and Initiator tRNA

Heterotrimeric a/eIF2αβγ (archaeal homologue of the eukaryotic translation initiation factor 2 with α, β and γ subunits) delivers charged initiator tRNA (tRNAi) to the small ribosomal subunit. In this work, we determined the structures of aIF2γ from the archaeon Sulfolobus solfataricus in the nucleotide-free and GDP-bound forms. Comparison of the free, GDP and Gpp(NH)p–Mg 2+ forms of aIF2γ revealed a sequence of conformational changes upon GDP and GTP binding. Our results show that the affinity of GDP to the G domain of the γ subunit is higher than that of Gpp(NH)p. In analyzing a pyrophosphate molecule binding to domain II of the γ subunit, we found a cleft that is very suitable for the acceptor stem of tRNA accommodation. It allows the suggestion of an alternative position for Met–tRNA i Met on the αγ intersubunit dimer, at variance with a recently published one. In the model reported here, the acceptor stem of the tRNAi is approximately perpendicular to that of tRNA in the ternary complex elongation factor Tu–Gpp(NH)p–tRNA. According to our analysis, the elbow and T stem of Met–tRNA i Met in this position should make extensive contact with the α subunit of aIF2. Thus, this model is in good agreement with experimental data showing that the α subunit of aIF2 is necessary for the stable interaction of aIF2γ with Met–tRNA i Met.

Role of the Transcription Factor ATF4 in the Anabolic Actions of Insulin and the Anti-anabolic Actions of Glucocorticoids

In most mammalian cells, insulin and glucocorticoids promote anabolism and catabolism, respectively. Whereas the opposing effects of insulin and glucocorticoids on catabolic gene expression have been explained at the molecular level, comparatively little is known about how these hormones alter anabolic gene expression. These studies identify ATF4 as an anabolic transcription factor that is repressed by glucocorticoids and induced by insulin. Insulin-mediated induction of ATF4 required the mammalian target of rapamycin complex 1, was required for the activation of a genetic program for the cellular uptake of essential amino acids and the synthesis of nonessential amino acids and aminoacyl-tRNAs, and was coupled to the repression of Foxo-dependent genes needed for protein and lipid catabolism. These results suggest that ATF4 plays a central role in hormonal regulation of amino acid and protein anabolism by coupling amino acid uptake and synthesis, as well as the generation of charged tRNAs, to mammalian target of rapamycin complex 1-mediated mRNA translation.

Differences between women who persist on weekly vs. monthly bisphosphonate therapy: results from the dash study (drivers of adherence to bisphosphonates)

The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth.

Tyrosyl-tRNA synthetase: the first crystallization of a human mitochondrial aminoacyl-tRNA synthetase.

Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA(Tyr) charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4(3)2(1)2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 A resolution. Complete data sets could be collected and led to structure solution by molecular replacement.

Single molecule fluorescence detection of BODIPY-FL molecules for monitoring protein synthesis

We have observed single DNA strands labeled with a small fluorescent molecule, BODIPY-FL, using objective based total internal fluorescence microscopy. In spite of its photobleaching, this small fluorophore is potentially useful for monitoring biochemical processes like protein synthesis because it can be efficiently incorporated into proteins by the ribosome through a charged epsilon-labeled fluorescent lysine tRNA. Using evanescent wave laser excitation at 488 nm, we have been able to observe single Bodipy molecules using integration times as low as 20 ms with a good signal to noise ratio, and for several images before photobleaching. We have measured the fluorescence decay due to photobleaching and have been able to lengthen it by a factor of 5 using oxygen scavenger systems.

Loss of a Universal tRNA Feature

tRNA(His) has thus far always been found with one of the most distinctive of tRNA features, an extra 5' nucleotide that is usually a guanylate. tRNA(His) genes in a disjoint alphaproteobacterial group comprising the Rhizobiales, Rhodobacterales, Caulobacterales, Parvularculales, and Pelagibacter generally fail to encode this extra guanylate, unlike those of other alphaproteobacteria and bacteria in general. Rather than adding an extra 5' guanylate posttranscriptionally as eukaryotes do, evidence is presented here that two of these species, Sinorhizobium meliloti and Caulobacter crescentus, simply lack any extra nucleotide on tRNA(His). This loss correlates with changes at the 3' end sequence of tRNA(His) and at many sites in histidyl-tRNA synthetase that might be expected to affect tRNA(His) recognition, in the flipping loop, the insertion domain, the anticodon-binding domain, and the motif 2 loop. The altered tRNA charging system may have affected other tRNA charging systems in these bacteria; for example, a site in tRNA(Glu) sequences was found to covary with tRNA(His) among alphaproteobacteria.

Crystal Structure of a Non-discriminating Glutamyl-tRNA Synthetase

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA Gln is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA Glu and tRNA Gln with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA Glu. Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS Tel ) in complex with glutamate at a resolution of 2.45 Å. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS Tth ). We confirm experimentally that GluRS Tel is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA Glu and of Glu-tRNA Gln. Anticodons of tRNA Glu ( 34C/UUC 36) and tRNA Gln ( 34C/UUG 36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS Tth by the residue Arg358. In ND-GluRS Tel this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA Gln to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.

Two conformations of a crystalline human tRNA synthetase–tRNA complex: implications for protein synthesis

Aminoacylation of tRNA is the first step of protein synthesis. Here, we report the co-crystal structure of human tryptophanyl-tRNA synthetase and tRNATrp. This enzyme is reported to interact directly with elongation factor 1alpha, which carries charged tRNA to the ribosome. Crystals were generated from a 50/50% mixture of charged and uncharged tRNATrp. These crystals captured two conformations of the complex, which are nearly identical with respect to the protein and a bound tryptophan. They are distinguished by the way tRNA is bound. In one, uncharged tRNA is bound across the dimer, with anticodon and acceptor stem interacting with separate subunits. In this cross-dimer tRNA complex, the class I enzyme has a class II-like tRNA binding mode. This structure accounts for biochemical investigations of human TrpRS, including species-specific charging. In the other conformation, presumptive aminoacylated tRNA is bound only by the anticodon, the acceptor stem being free and having space to interact precisely with EF-1alpha, suggesting that the product of aminoacylation can be directly handed off to EF-1alpha for the next step of protein synthesis.

Model of gene expression regulation in bacteria via formation of RNA secondary structures

A model was proposed for the classical attenuating mRNA regulation of gene expression via transcription termination. The model is based on the concept of secondary structure macrostates in the RNA regulatory region between the ribosome and RNA polymerase, utilizes resonant equations for estimating the deceleration of RNA polymerase by a set of hairpins located in this RNA region, and takes into account views on the initiation and elongation of transcription and translation. Special attention was paid to selecting the model parameters. To test the model, computations were performed to estimate, in particular, the probability of translation termination as dependent on the charged tRNA concentration and the amino acid concentration for several regulatory regions of the bacterial genome (as exemplified by trpE of Streptomyces spp., Bradyrhizobium japonicum, and Escherichia coli). Analysis was performed with different values of three parameters isolated as major ones. The resulting dependences agreed with the available experimental data, including those characterizing an enzymatic activity as dependent on the amino acid concentration in a culture (e.g., the anthranylate synthase activity as dependent on the tryptophan concentration in S. venezuelae). The following possible application was proposed for the model. Attenuating regulation is usually predicted on the basis of multiple sequence alignment, which requires several sequences. With the model, an individual sequence can be analyzed with proper parameters to generate a concentration-enzymatic activity curve. The curve characteristic of attenuation or its absence provides an additional argument for the presence or absence of attenuation.

A patient with pancreatitis, anaemia and an intrathoracic tumour

<h3>Abstract</h3> A label-free procedure to identify single amino acids (AAs) for protein sequencing is developed in theory and simulated in part. A terminal AA cleaved from a protein/peptide, a tRNA, its cognate amino-acyl tRNA synthetase (AARS), and adenosine triphosphate (ATP) are brought together in a container where tRNA, if cognate, gets charged with AA and adenosine monophosphate (AMP) is released. The released AMP (and any free AA and ATP molecules) filters into the <i>cis</i> chamber of an adjoining electrolytic cell (e-cell) from where they pass through a nanopore into the <i>trans</i> chamber. Addition of NaOH to the container deacylates the tRNA if it is charged. The resulting free AA passes into the <i>cis</i> chamber of the e-cell, translocates into <i>trans</i>, and causes a current blockade; AA is immediately known from the identity of the tRNA (the two are cognate). If the tRNA is not charged there is no AA bound to it so AA remains unidentified. In this approach there is no need to distinguish among the 20 AAs by blockade size; it suffices to distinguish blockades from noise: thus a high-precision analog measurement has been transformed into a low-precision binary one. Identification is accurate because of tRNA superspecificity (the tRNA charging error rate is &lt; 1/350); parallel execution with 20 different tRNAs can identify AA in one cycle. This is a <i>de novo</i> method in which no prior information about the protein is used or needed.