Functional Substitution of a Eukaryotic Glycyl-tRNA Synthetase with an Evolutionarily Unrelated Bacterial Cognate Enzyme

Two oligomeric types of glycyl-tRNA synthetase (GlyRS) are found in nature: a α₂ type and a α₂β₂ type. The former has been identified in all three kingdoms of life and often pairs with tRNA^Gly that carries an A73 discriminator base, while the latter is found only in bacteria and chloroplasts and is almost always coupled with tRNA^Gly that contains U73. In the yeast Saccharomyces cerevisiae, a single GlyRS gene, GRS1, provides both the cytoplasmic and mitochondrial functions, and tRNA^Gly isoacceptors in both compartments possess A73. We showed herein that Homo sapiens and Arabidopsis thaliana cytoplasmic GlyRSs (both α₂-type enzymes) can rescue both the cytoplasmic and mitochondrial defects of a yeast grs1 ^- strain, while Escherichia coli GlyRS (a α₂β₂-type enzyme) and A. thaliana organellar GlyRS (a (αβ)₂-type enzyme) failed to rescue either defect of the yeast mull allele. However, a head-to-tail αβ fusion of E. coli GlyRS effectively supported the mitochondrial function. Our study suggests that a α₂-type eukaryotic GlyRS may be functionally substituted with a α₂β₂-type bacterial cognate enzyme despite their remote evolutionary relationships.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

Calcium Ion Impedes Translation Initiation at the Synapse

Abstract: Stimulation of synaptoneurosome suspensions by the neurotransmitter glutamate gives rise to rapid loading of ribosomes onto mRNA and increased incorporation of amino acids into trichloroacetic acid-precipitable polypeptides. Metabotropic glutamate receptors (mGluRs) are responsible for this effect. Although simultaneous Ca²⁺ entry and mGluR stimulation do not change the response, entry of Ca²⁺ 30 s or 3 min before mGluR stimulation markedly depresses the polyribosomal loading. Either NMDA or ionophore (A23187) produces the depression. A calmodulin antagonist, W7, alleviates the effect, suggesting that inactivation of phospholipase A₂ by calcium-calmodulin-dependent kinase II is partially responsible for the phenomenon. Thus, interaction between different classes of glutamate receptors affects the control of protein translation at the synapse. This effect may partially explain recent observations of negative interactions between receptor classes in induction of long-term potentiation.     

Cocrystal Structures of Glycyl-tRNA Synthetase in Complex with tRNA Suggest Multiple Conformational States in Glycylation

Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charges tRNA molecules with cognate amino acids for protein synthesis. Glycyl-tRNA synthetase (GlyRS) is one of the most intriguing aminoacyl-tRNA synthetases due to its divergent quaternary structure and abnormal charging properties. In the past decade, mutations of human GlyRS (hGlyRS) were also found to be associated with Charcot-Marie-Tooth disease. However, the mechanisms of traditional and alternative functions of hGlyRS are poorly understood due to a lack of studies at the molecular basis. In this study we report crystal structures of wild type and mutant hGlyRS in complex with tRNA and with small substrates and describe the molecular details of enzymatic recognition of the key tRNA identity elements in the acceptor stem and the anticodon loop. The cocrystal structures suggest that insertions 1 and 3 work together with the active site in a cooperative manner to facilitate efficient substrate binding. Both the enzyme and tRNA molecules undergo significant conformational changes during glycylation. A working model of multiple conformations for hGlyRS catalysis is proposed based on the crystallographic and biochemical studies. This study provides insights into the catalytic pathway of hGlyRS and may also contribute to our understanding of Charcot-Marie-Tooth disease.     

Model of the Complex of the Human Glycyl-tRNA Synthetase Anticodon-Binding Domain with IRES I Fragment

The currently available structural information is insufficient for a detailed analysis of interactions between human glycyl-tRNA synthetase (GARS) and enterovirus IRESs. At the same time, this information is required in order to understand how this IRES trans-acting factor (ITAF) functions during viral mRNA translation, which is in turn crucial for the development of direct-action antiviral agents. In this paper, a theoretical model of the complex between a cadicivirus A IRES fragment and the anticodon-binding domain of human GARS is constructed using molecular dynamics simulation based on all of the available structural and biochemical data. The proposed model enables the structural interpretation of the previously obtained biochemical data.     

Large Conformational Changes of Insertion 3 in Human Glycyl-tRNA Synthetase (hGlyRS) during Catalysis

Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great research interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNA^Gly respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a “switch” and fully opens to allow tRNA to bind in a cross-subunit fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases).     

Melanotropin as a Potential Regulator of Pigment Pattern Formation in Embryonic Skin

Frozen tissue sections of developing axolotl embryos were labeled by indirect immunofluorescence with anti-alpha-MSH. Anti-MSH immunoreactivity is first detectable in embryos when neural crest cells are migrating from the neural tube. Antibody labeling is visible around the lateral and ventral edges of the neural tube and in the embryonic ectoderm. As development progresses, the amount of labeling increases greatly, particularly in developing ectoderm. Western blots of soluble proteins extracted from various developmental stages of axolotl embryo ectoderm reveal that MSH activity is associated directly with several high molecular weight components that may be part of the embryonic extracellular matrix. Thus, we suggest that melanotropin activity is present in embryonic axolotl skin, is associated with the extracellular matrix, and is thereby in a position to play a supportive and/or directive role in the establishment of embryonic pigment patterns.     

Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation

Background

Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5′ or 3′ extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5′end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle.

Principal Findings

We have demonstrated that PTB interacts with at least two binding sites within the 5′end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication.

Conclusions

Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA.     

不依赖甲硫氨酸的翻译起始

Ｓａｓａｋｉ等在ＰｒｏｃＮａｔｌＡｃａｄＳｃｉＵＳＡ 2 0 0 0年第 4期上报道了一种不依赖甲硫氨酸的翻译起始方式[1] 。ＰＳＩＶ (Ｐｌａｕｔｉａｓｔａｌｉｉｎｔｅｓｔｉｎｅｖｉｒｕｓ)是一种昆虫ＲＮＡ病毒 ,属蟋蟀麻痹样病毒组 (Ｃｒｉｃｋｅｔｐａｒａｌｙｓｉｓ ｌｉｋｅｖｉｒｕｓｅｓ) ,为正链ＲＮＡ病毒。属于该组的还有ＤＣＶ、ＲｈＰＶ、ＨｉＰＶ等。该组病毒的理化性质与哺乳动物的小ＲＮＡ病毒 (ｐｉｃｏｒｎａｖｉｒｕｓ)相似 ,但基因图 1　 (Ａ)ＰＳＩＶ基因组结构 ;(Ｂ)预测的外壳蛋白编码区上游的茎环结构组织不同。…     

An Insertion Peptide in Yeast Glycyl-tRNA Synthetase Facilitates both Productive Docking and Catalysis of Cognate tRNAs

The yeast Saccharomyces cerevisiae possesses two distinct glycyl-tRNA synthetase (GlyRS) genes: GRS1 and GRS2. GRS1 is dually functional, encoding both cytoplasmic and mitochondrial activities, while GRS2 is dysfunctional and not required for growth. The protein products of these two genes, GlyRS1 and GlyRS2, are much alike but are distinguished by an insertion peptide of GlyRS1, which is absent from GlyRS2 and other eukaryotic homologues. We show that deletion or mutation of the insertion peptide modestly impaired the enzyme''s catalytic efficiency in vitro (with a 2- to 3-fold increase in K_m and a 5- to 8-fold decrease in k_cat). Consistently, GRS2 can be conveniently converted to a functional gene via codon optimization, and the insertion peptide is dispensable for protein stability and the rescue activity of GRS1 at 30°C in vivo. A phylogenetic analysis further showed that GRS1 and GRS2 are paralogues that arose from a gene duplication event relatively recently, with GRS1 being the predecessor. These results indicate that GlyRS2 is an active enzyme essentially resembling the insertion peptide-deleted form of GlyRS1. Our study suggests that the insertion peptide represents a novel auxiliary domain, which facilitates both productive docking and catalysis of cognate tRNAs.     

Regulation of Nitrogen Fixation in Klebsiella pneumoniae: Evidence for a Role of Glutamine Synthetase as a Regulator of Nitrogenase Synthesis

Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.     

Crystal Structures and Biochemical Analyses Suggest a Unique Mechanism and Role for Human Glycyl-tRNA Synthetase in Ap4A Homeostasis

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed.Aminoacyl-tRNA synthetases (AARSs)⁴ are considered to be among the earliest proteins to have emerged during evolution. As a family of typically 20 members (one for each amino acid), AARSs catalyze the first step of protein synthesis by linking each amino acid onto the 3′-end of its cognate tRNA harboring the trinucleotide anticodon. Through evolution, the role of AARSs has also been broadened with expanded functions (reviewed in Refs. 1 and 2). These expanded functions often involve direct interaction partners. For example, human tyrosyl-tRNA synthetase interacts with chemokine receptor CXCR1 to induce cell migration (3); human glutaminyl-tRNA synthetase interacts with ASK1 to regulate apoptosis (4); human tryptophanyl-tRNA synthetase interacts with VE-cadherin to inhibit angiogenesis (5); human lysyl-tRNA synthetase interacts with the Gag protein of human immunodeficiency virus to facilitate viral assembly (6); and human glutamyl-prolyl-tRNA synthetase interacts with L13a and glyceraldehyde-3-phosphate dehydrogenase to form the GAIT complex for translational silencing to regulate inflammation (7). However, functional expansion also can be achieved indirectly via reaction products of AARSs. As examples, Lys-tRNA^Lys and Ala-tRNA^Ala are used to aminoacylate cytoplasmic membrane phosphatidylglycerol of Staphylococcus aureus and Pseudomonas aeruginosa, respectively, to enhance drug resistance in these microorganisms (8).In addition to tRNA aminoacylation, the majority of AARSs have the capacity to catalyze a side reaction to form diadenosine oligophosphates (ApnA) in the absence of cognate tRNA (9). These reactions of AARS are the most well known sources of ApnA in vivo (10). ApnA are made up of two adenosine moieties linked at the 5′-end of the ribose by a chain of two to six phosphates. In the 4 decades following the discovery of these molecules by Zamecnik et al. (10), ApnA have been linked to highly diverse physiological effects in prokaryotic and eukaryotic cells, including various types of mammalian cells and tissues, and to assorted functions associated with the nucleus, membrane receptors, and activities in the cytoplasm (reviewed in Refs. 11 and 12). The concentrations of ApnA molecules in vivo respond to numerous factors, including cell proliferation status, glucose level, heat shock, oxidative stress, and interferon stimulation. They have emerged as extracellular and intracellular signaling molecules (as pleiotropically acting “alarmones” (13) and second messengers (14)) implicated in the maintenance and regulation of vital cellular functions.The aminoacylation reaction proceeds in two steps. First, the amino acid is activated by condensation with ATP to form aminoacyl-AMP, the enzyme-bound intermediate. The aminoacyl moiety is then transferred to the 3′-end of the cognate tRNA. When tRNA is absent, the enzyme-bound aminoacyl-AMP can be attacked by the γ-phosphate of a second ATP molecule to form diadenosine tetraphosphate (Ap4A), the most common diadenosine oligophosphate produced by a tRNA synthetase (see Fig. 1A). The presence of tRNA in most cases inhibits Ap4A synthesis (11). Therefore, a subgroup of tRNA synthetases that requires tRNA as cofactor for synthesis of aminoacyl-AMP is not capable of producing Ap4A. This group includes tRNA synthetases that are specific for arginine, glutamine, and glutamic acid and an unusual class I lysyl-tRNA synthetase (LysRS).Open in a separate windowFIGURE 1.Amino acid-independent synthesis of Ap4A by human GlyRS. A, conventional mechanism for synthesis of Ap4A by a tRNA synthetase. The first step of the reaction involves the generation of an enzyme-bound aminoacyl-AMP (aa-AMP), which is then attacked either by cognate tRNA to form aminoacyl-tRNA or by ATP to form Ap4A. B, mechanism used by human GlyRS to produce Ap4A by direct condensation of two ATPs. C–E, synthesis of Ap4A by GlyRS, LysRS, and TyrRS, respectively, in the presence (●) and absence (○) of cognate amino acid. Amounts of Ap4A produced were quantitated from the TLC sheets and are plotted on the right. GlyRS activity was measured in triplicate, and the plotted values reflect the mean ± S.E.Although the amino acid recycles, the above mechanism requires the presence of the amino acid for the production of Ap4A via the aminoacyl-AMP intermediate. Using biochemical analyses and determinations of co-crystal structures, we demonstrate in this work that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs in the absence of glycine. Thus, the mechanism for GlyRS to synthesize Ap4A is decoupled from aminoacylation. Furthermore, GlyRS is likely to be the only synthetase that produces Ap4A by this mechanism. Our results raise the possibility that GlyRS plays a special role in Ap4A homeostasis.     

真核生物蛋白质翻译的内部起始机制

蛋白质翻译过程中,翻译的起始步骤是非常重要的．真核生物的翻译起始主要是通过依赖帽子结构的扫描机制进行的．近几年在翻译的研究工作中发现,在一些动物病毒中,蛋白质合成通过一种不同于扫描机制的内部起始机制起始翻译．用内部起始机制翻译的mRNA的5′端非翻译区有一个相对保守的结构,它在内部起始过程中具有重要作用,一些特异的蛋白质因子能够促进在特定位点起始翻译．     

Structure-Function Relation of Phospholamban: Modulation of Channel Activity as a Potential Regulator of SERCA Activity

Phospholamban (PLN) is a small integral membrane protein, which binds and inhibits in a yet unknown fashion the Ca²⁺-ATPase (SERCA) in the sarcoplasmic reticulum. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a low unitary conductance. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 Å for this channel. This value is similar to that reported for the central pore in the structure of the PLN pentamer. Hence the PLN pentamer, which is in equilibrium with the monomer, is the most likely channel forming structure. Reconstituted PLN mutants, which either stabilize (K27A and R9C) or destabilize (I47A) the PLN pentamer and also phosphorylated PLN still generate the same unitary conductance of the wt/non-phosphorylated PLN. However the open probability of the phosphorylated PLN and of the R9C mutant is significantly lower than that of the respective wt/non-phosphorylated control. In the context of data on PLN/SERCA interaction and on Ca²⁺ accumulation in the sarcoplasmic reticulum the present results are consistent with the view that PLN channel activity could participate in the balancing of charge during Ca²⁺ uptake. A reduced total conductance of the K⁺ transporting PLN by phosphorylation or by the R9C mutation may stimulate Ca²⁺ uptake in the same way as an inhibition of K⁺ channels in the SR membrane. The R9C-PLN mutation, a putative cause of dilated cardiomyopathy, might hence affect SERCA activity also via its inherent low open probability.     

Site-Directed Mutagenesis of a Saccharomyces Cerevisiae Mitochondrial Translation Initiation Codon

We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA. The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII). Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide. Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA. Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA. Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA. Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets. We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.     

mRNA翻译起始区的结构改变对几个外源基因翻译的影响

为观察ｍＲＮＡ翻译起始区结构与基因表达的关系,利用密码子的简并性,在不改变表达产物氨基酸序列的前提下定点突变几个外源基因的５′端若干位点,使基佤表达载体重组后转录形成的ｍＲＮＡ翻译起始区结构发生改变。经ＳＤＳ－ＰＡＧＥ等分析证实这些改变大大提高了外源基因的表达水平,ＲＮＡｄｏｔｂｌｏｔ表明突变与非突变基因转录水平差别不大,表达水平的提高主要由于翻译效率的提高,ｍＲＮＡ翻译起始区二级结构预测提示其生