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1.
It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype onEscherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNAGln: GlnRS:ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3 end of tRNAGln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischargesupF tRNATyr considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguishsupE tRNA CUA Gln from tRNA 2 Gln considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher Km for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.  相似文献   

2.
3.
Discrimination of tRNAGln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNAGln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNAGln. Our results demonstrate that in addition to the anticodon-binding domain, tRNAGln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.  相似文献   

4.
The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNAGln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNAGln and tRNAGlu with glutamate. This ancient GluRS also separately differentiated to exclude tRNAGln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNAGln and tRNAGlu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.  相似文献   

5.
Aminoacylation of transfer RNAGln (tRNAGln) is performed by distinct mechanisms in different kingdoms and represents the most diverged route of aminoacyl-tRNA synthesis found in nature. In Saccharomyces cerevisiae, cytosolic Gln-tRNAGln is generated by direct glutaminylation of tRNAGln by glutaminyl-tRNA synthetase (GlnRS), whereas mitochondrial Gln-tRNAGln is formed by an indirect pathway involving charging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNAGln amidotransferase. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for its yeast homologue in vivo. We report herein that the same fusion enzyme, upon being imported into mitochondria, substituted the indirect pathway for Gln-tRNAGln synthesis as well, despite significant differences in the identity determinants of E. coli and yeast cytosolic and mitochondrial tRNAGln isoacceptors. Fusion of Arc1p to the bacterial enzyme significantly enhanced its aminoacylation activity towards yeast tRNAGln isoacceptors in vitro. Our study provides a mechanism by which trans-kingdom rescue of distinct pathways of Gln-tRNAGln synthesis can be conferred by a single enzyme.  相似文献   

6.
Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNAGln formation. To analyze the peculiarities of the structure–function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3Å and a docking model of the synthetase complexed to tRNAGln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNAGln are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNAGln recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNAGln acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.  相似文献   

7.
The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNAGln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNAGlu and Glu-tRNAGln. The Glu-tRNAGln is then converted to Gln-tRNAGln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNAGlu and tRNAGln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNAGlu/tRNAGln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNAGln complex reveals the structural determinants responsible for specific tRNAGln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.  相似文献   

8.
This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNAGln. Temperature-sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNAGln: GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNAGln species were synthesized with either a 2′-deoxy AMP or 3′-deoxy AMP as their 3′-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PPi exchange activity established the esterification of glutamine to the 2′-hydroxyl of the terminal adenosine: there is no glutaminylation of the 3′-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNAGln are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed.  相似文献   

9.
Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2. Like a canonical GluRS, GluRS1 aminoacylates tRNAGlu1 and tRNAGlu2. In contrast, GluRS2 only misacylates tRNAGln to form Glu-tRNAGln. It is not clear how GluRS2 achieves specific recognition of tRNAGln while rejecting the two H. pylori tRNAGlu isoacceptors. Here, we show that GluRS2 recognizes major identity elements clustered in the tRNAGln acceptor stem. Mutations in the tRNA anticodon or at the discriminator base had little to no impact on enzyme specificity and activity.  相似文献   

10.
11.
In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNAGln is primarily synthesized by first forming Glu-tRNAGln, followed by conversion to Gln-tRNAGln by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNAGln and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNAGln. These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNAGln, consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNAGln synthesis.  相似文献   

12.
In order to isolate the gene for amber suppressor su+2 (SupE) in Escherichia coli, a non-defective su+2-transducing phage lambda was isolated in three steps: first, deletion derivatives of F′su+2 gal (λ) were selected, linking su+2 to the right-hand prophage attachment site, attλPB′; second, these F′-factors were relysogenized by λ and defective transducing phages, λdsu+2, were produced by induction; and third, non-defective λpsu+2 transducing phages were produced by recombination of λdsu+2 isolates with λ. Upon infection by λpsu+2, the production of transferRNAs accepting glutamine and methionine was markedly stimulated. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNA2Gln, mutant tRNA2Gln and tRNAmMet. The mutant tRNA2Gln carried a singlebase alteration from G to A at the 3′-end of the anticodon. The production of tRNA1Gln was not stimulated by the infection of λpsu+2. We conclude that the wild-type allele of su+2 (SupE) is the structural gene for tRNA2Gln, and the su+2 amber suppressor was derived by a single base mutation, changing the anticodon from CUG to CUA, in one of the multi-copy genes for tRNA2Gln. The fact that λpsu+2 also induces the production of tRNAmMet suggests that this tRNA is encoded in the same chromosomal region of E. coli as is tRNA2Gln.  相似文献   

13.
The number of gene copies for tRNA2Gln in λpsu+2 was determined by genetic and biochemical studies. The transducing phage stimulates the production of the su+2 (amber suppressor) and su°2 glutamine tRNAs and methionine tRNAm. When the su+2 amber suppressor was converted to an ochre suppressor by single-base mutation, the phage stimulated ochre-suppressing tRNA2Gln, instead of the amber-suppressing tRNA2Gln. From the transducing phage carrying the ochre-suppressing allele, strains carrying both ochre and amber suppressors were readily obtainable. These phages stimulated both ochre-suppressing and amber-suppressing tRNA2Gln, but not the non-suppressing form. We conclude that the original transducing phage carries two tRNA2Gln genes, one su+2 and one su°2. The transducing phage carrying two suppressors, ochre and amber, segregates one-gene derivatives that encode only one or the other type of suppressor tRNA. These derivatives apparently arise by unequal recombination involving the two glutamine tRNA genes in the parental phage. This segregation is not accompanied by the loss of the tRNAmMet gene. Based on these results, it is suggested that Escherichia coli normally carries in tandem two identical genes specifying tRNA2Gln at 15 minutes on the bacterial chromosome. su+2 mutants may arise by single-base mutations in the anticodon region of either of these two, leaving the other intact. By double mutations, tRNA2Gln genes could also become ochre suppressors. A tRNAmMet gene is located near, but not between, these two tRNA2Gln genes.  相似文献   

14.
The three major glutamine tRNAs of Tetrahymena thermophila were isolated and their nucleotide sequences determined by post-labeling techniques. Two of these tRNAsGln show unusual codon recognition: a previously isolated tRNAGlnUmUA and a second species with CUA in the anticodon (tRNAGlnCUA). These two tRNAs recognize two of the three termination codons on natural mRNAs in a reticulocyte system. tRNAGlnUmUA reads the UAA codon of α-globin mRNA and the UAG codon of tobacco mosaic virus (TMV) RNA, whereas tRNAGlnCUA recognizes only UAG. This indicates that Tetrahymena uses UAA and UAG as glutamine codons and that UGA may be the only functional termination codon. A notable feature of these two tRNAsGln is their unusually strong readthrough efficiency, e.g. purified tRNAGlnCUA achieves complete readthrough over the UAG stop codon of TMV RNA. The third major tRNAGln of Tetrahymena has a UmUG anticodon and presumably reads the two normal glutamine codons CAA and CAG. The sequence homology between tRNAGlnUmUG and tRNAGlnUmUA is 81%, whereas that between tRNAGlnCUA and tRNAGlnUmUA is 95%, indicating that the two unusual tRNAsGln evolved from the normal tRNAGln early in ciliate evolution. Possible events leading to an altered genetic code in ciliates are discussed.  相似文献   

15.
Synthesis of T4 tRNAGln depends on normal levels of Escherichiacoli ribonuclease III. Infection of cell strains carrying a mutation in the gene for this enzyme resulted in severe depression in tRNAGln production, as revealed by chemical and suppressor tRNA analyses. The remaining seven T4 tRNAs were synthesized in the mutant cells. The requirement of ribonuclease III for synthesis of tRNAGln points to an essential cleavage by the enzyme of a precursor RNA containing tRNAGln.  相似文献   

16.
We have constructed a model of the complex between tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and tRNATyr by successive cycles of predictions, mutagenesis of TyrRS and molecular modeling. We confront this model with data obtained independently, compare it to the crystal structures of other complexes and review recent data on the discrimination between tRNAs by TyrRS. Comparison of the crystal structures of TyrRs and GlnRS, both of which are class I synthetases, and comparison of the identity elements of tRNATyr and tRNAGln indicate that the two synthetases bind their cognate tRNAs differently. The mutagenesis data on tRNATyr confirm the model of the TyrRS:tRNATyr complex on the following points. TyrRS approaches tRNATyr on the side of the variable loop. The bases of the first three pairs of the acceptor stem are not recognized. The presence of the NH2 group in position C6 and the absence of a bulky group in position C2 are important for the recognition of the discriminator base A73 by TyrRS, which is fully realized only in the transition state for the acyl transfer. The anticodon is the major identity element of tRNATyr. We have set up an in vivo approach to study the effects of synthetase mutations on the discrimination between tRNAs. Using this approach, we have shown that residue Glul52 of TyrRS acts as a purely negative discriminant towards non-cognate tRNAs, by electrostatic and steric repulsions. The overproductions of the wild type TyrRSs from E coli and B stearothermophilus are toxic to E coli, due to the mischarging or the non-productive binding of tRNAs. The construction of a family of hybrids between the TyrRSs from E coli and B stearothermophilus has shown that their sequences and structures have remained locally compatible through evolution, for holding and function, in particular for the specific recognition and charging of tRNATyr.  相似文献   

17.
We isolated several mutants with nucleotide substitutions in alanine tRNA (tRNAAla) that resulted in glutamine tRNA (tRNAGli) acceptor identity in Escherichia coli. These substitutions were in three regions of tRNA structure not previously associated with tRNAGln acceptor identity. Only the phosphate-sugar backbone moieties of these nucleotides interact with the enzyme in the previously determined X-ray crystal structure of the complex between tRNAGln and glutaminyl-tRNA synthetase. We conclude that these sequence-dependent phosphate-sugar backbone interactions contribute to tRNAGln identity, and argue that the interactions help communicate enzyme recognition of the anticodon to the acceptor end of the tRNA and the catalytic center of the enzyme.  相似文献   

18.
The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNAGln, that AspRS is nondiscriminating, and that its Asp-tRNAAsn product is transamidated by AdT. On the other hand, tRNAGln is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.  相似文献   

19.
Chemically synthesized genes encodingEscherichia coli tRNA 1 Leu and tRNA 2 Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A260, respectively: the content of tRNA 1 Leu was 50% of total tRNA from MT-Leu1, while that of tRNA 2 Leu was 30% of total tRNA from MT-Leu2. Both tRNALeus from their rotal tRNs were fractionated to 1 600 pmol/A260 after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNALeu catalyzed by leucyl-tRNA synthetase were determined.  相似文献   

20.
Yeast Saccharomyces cerevisiae MTO2, MTO1, and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of carboxymethylaminomethyl (cmnm)5s2U34 in mitochondrial tRNALys, tRNAGlu, and tRNAGln. In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm5 group (cmnm5U34), while Mto2p is responsible for the 2-thiouridylation (s2U34) of these tRNAs. Previous studies showed that partial modifications at U34 in mitochondrial tRNA enabled mto1, mto2, and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1, and MSS1 genes by using the mto2, mto1, and mss1 single, double, and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol but not on medium containing glucose. Interestingly, there were no detectable levels of nine tRNAs including tRNALys, tRNAGlu, and tRNAGln in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1, and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA and 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U34 of tRNALys, tRNAGlu, and tRNAGln, caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm5U34 by the mto1 or mss1 mutation. The complete loss of modifications at U34 of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA, and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory-deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U34 of tRNALys, tRNAGlu, and tRNAGln in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function.  相似文献   

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