Yeast aspartyl-tRNA synthetase residues interacting with tRNAAsp identity bases connectively contribute to tRNAAsp binding in the ground and transition-state complex and discriminate against non-cognate tRNAs

International audienceCrystallographic studies of the aspartyl-tRNA synthetase-tRNAAsp complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNAAsp. Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNAAsp recognition and acylation. The results showed that contacts with the acceptor A of tRNAAsp by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNAAsp. Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state. These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis. This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site. This signal might be conveyed via tRNAAspas suggested by the observed conformational change of this molecule upon interaction with AspRS. From binding free energy values corresponding to the different AspRS-tRNAAsp interaction domains, it might be concluded that upon complex formation, the anticodon interacts first. Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNAAsp and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs

Assembly And Function Of Macromolecular Complexes For Accurate Trna Aminoacylation In Helicobacter Pylori

ABSTRACT ASSEMBLY AND FUNCTION OF MACROMOLECULAR COMPLEXES FOR ACCURATE TRNA AMINOACYLATION IN HELICOBACTER PYLORI by GAYATHRI SILVA January 2014 Advisor: Dr. Tamara L. Hendrickson Major: Chemistry (Biochemistry) Degree: Doctor of Philosophy Abstract The aminoacylation of tRNA is a critical step in maintaining the accuracy of the genetic code. Many microorganisms are missing one or more aminoacyl tRNA synthetases (aaRSs) and rely on indirect pathways to produce certain aa–tRNAs. In Helicobacter pylori (H. pylori), the genes encoding both asparaginyl tRNA synthetase (AsnRS) and glutaminyl tRNA synthetase (GlnRS) are missing and the organism consequently relies on the indirect pathway for the synthesis of Asn–tRNAAsn and Gln–tRNAGln. The first step of indirect synthesis of Asn–tRNAAsn involves misacylation of tRNAAsn by non–discriminating aspartyl tRNA synthetase (ND–AspRS) to produce Asp–tRNAAsn. Next, the misacylated tRNA is converted to Asn–tRNAAsn by Asp–tRNAAsn/Glu–tRNAGln amidotransferase (Asp/Glu–AdT) to produce Asn–tRNAAsn. Gln–tRNAGln is produced vial an analogous process, relying on a misacylating glutamyl tRNA synthetase, GluRS2 and AdT. Including H. pylori, organisms that indirectly synthesize Asn–tRNAAsn and Gln–tRNAGln require a mechanism for the efficient delivery of both misacylated tRNAs from the two misacylating enzymes to the amidotransferase enzyme. This delivery mechanism should ensure the stability of the aminoacyl ester bond and prevent translational errors. Some bacteria, like Thermus thermophilus (T. thermophilus), utilize a tRNA–dependent ribonucleoprotein complex (RNC) called the transamidosome. The T. thermophilus transamidosome contains AdT, tRNAAsn, and an archaeal ND–AspRS. This Asn–transamidosome traps misacylated Asp–tRNAAsn until it has been converted to Asn–tRNAAsn by AdT. Similarly, the thermophilic archaeon Methanothermobacter thermoautotrophicus utilizes a Gln–transamidosome for the synthesis of Gln–tRNAGln. This complex consists of ND–GluRS, tRNAGln and GatDE. (GatDE is a heterodimeric homolog of AdT.) In contrast to T. thermophilus, H. pylori utilizes a bacterial ND–AspRS that has an extra domain that could sterically prevent transamidosome assembly. H. pylori also requires AdT for conversion of both Asp–tRNAAsn and Glu–tRNAGln into their cognate aa–tRNAs. In fact the H. pylori Asn– and Gln–transamidosomes were not stably isolated in vitro, suggesting a requirement for an alternative mechanism. We describe the first characterization of a novel protein called Hp0100 in H. pylori, Hp0100 is required for the assembly of a stable, tRNA–independent Asn–transamidosome, consisting of ND–AspRS, AdT and Hp0100. Hp0100 enhances the capacity of AdT to convert Asp–tRNAAsn into Asn–tRNAAsn and Glu–tRNAGln into Gln–tRNAGln but has minimal effect on ND–AspRS function. We discovered that Hp0100 is an ATPase which contains two distinct ATPase active sites that are activated by either of the two misacylated tRNAs, Glu–tRNAGln or Asp–tRNAAsn. The first ATP binding motif shares sequence similarity to adenine nucleotide alpha hydrolase–like (AANH–like) ATP binding motif superfamily. Surprisingly, mutations in this domain only disrupted the Glu–tRNAGln induced ATPase activity; the Asp–tRNAAsn activity was less affected (∼;50% decrease). The second motif shares sequence homology to the P–loop ATP binding motif. In contrast to AANH, P–loop mutations disrupted Asp–tRNAAsn induced ATPase activity but Glu–tRNAGln catalyzed ATPase activity was only partially reduced (∼;50% decrease). These results revealed that there are probably two mutually exclusive ATPase motifs in Hp0100 that are separately activated by Asp–tRNAAsn and Glu–tRNAGln. In addition, our mutagenesis studies also revealed the requirement of each ATPase motif for the corresponding acceleration in the rate of AdT transamidation of either Asp–tRNAAsn or Glu–tRNAGln. Overall, our results highlight the importance of the novel ATPase Hp0100, for the indirect biosynthesis of Asn–tRNAAsn and Gln–tRNAGln in H. pylori. H. pylori is an obligate human pathogen responsible for causing stomach ulcers and cancer. Its clade (the ε–proteobacteria) includes several human enteric pathogens like Campylobacter jejuni (C. jejuni) that cause other deleterious health problems in humans. Here we describe a unique mechanism used by this clade to ensure accuracy during indirect tRNA aminoacylation. Elucidation of the mechanisms used by other organisms holds potential for the development of a greater understanding of bacterial phylogenetics, speciation, and the identification of novel, clade specific targets for new antibiotics

Recognition of valine transfer RNA by valyl-tRNA synthetase from E coli: 19F NMR and aminoacylation studies involving wild type and mutant forms of 5-fluorouracil-substituted tRNAVal

The structural basis for the recognition of valine transfer RNA (tRNA[superscript] Val) by valyl-tRNA synthetase (VRS) from E. coli has been investigated by [superscript]19F NMR spectroscopy, aminoacylation kinetics and the technique or site-directed mutagenesis. The accomplishments presented here entail (1) the development of protocols for the synthesis of wild type and mutant tRNA[superscript] Val having 5-fluorouracil in place of uracil, (2) the direct assignment of [superscript]19F NMR peaks to specific FUra residues in tRNA[superscript] Val, and (3) the role played by specific bases in the recognition of tRNA[superscript] Val by its cognate synthetase;For in vitro synthesis of FUra-substituted tRNA[superscript] Val, plasmid pVAL119-21, combining a synthetic E. coli tRNA[superscript] Val gene, along with the T7 RNA promoter, was constructed. The synthetic tRNA gene has incorporated at its 3[superscript]\u27 terminus the restriction site for BstN1. Runoff transcription by T7 RNA polymerase of the BstN1 digested plasmid yields tRNA[superscript] Val which has no minor bases. The tRNA transcript, purified by HPLC, has a valine acceptance activity of 1200-1300 pmole/A[subscript]260;The [superscript]19F NMR spectra of the transcribed and native (FUra)tRNA[superscript] Val are identical except for slight differences in the chemical shifts of resonances in the central region of the spectrum (peaks E and F). Comparison of [superscript]19F NMR spectra of wild type and mutant forms of (FUra)tRNA[superscript] Val leads to the assignment of peaks B, C, E, F, H, K and L to fluorouracil 64, 59, 47, 33, 34, 29 and 67 respectively;Aminoacylation kinetic studies show that most of the mutant tRNAs could be aminoacylated by VRS. However, replacement of C36 with A, G, or U results in 150-1000 fold decrease in V[subscript] max/K[subscript] m. Mutants C34 and G41 also have lower V[subscript] max/K[subscript] m values. Addition of VRS induces several changes in the [superscript]19F NMR spectrum of (FUra) tRNA[superscript] Val. Intensity at 5 ppm increases, while the signal at 4 ppm (peak G/H) loses intensity. One peak shifts downfield from the cluster around 3 ppm. These results suggest an involvement of the anticodon in the recognition of tRNA[superscript] Val by VRS

Stability of the Complex between Yeast Seryl-tRNA Synthetase and tRNASer under Different Electrophoretic Conditions

Noncovalent interactions of yeast homodimeric seryl-tRNA synthetase (SerRS) and cognate tRNASer were studied by the gel mobility shift assay and zone-interference gel electrophoresis performed under the same binding and electrophoretic conditions. Purified tRNASer as well as total yeast tRNA were applied as ligands. In the absence of Mg2+, SerRS:(tRNASer)1 noncovalent complex was detected only by zone-interference gel electrophoresis. Kd values determined in the presence and absence of Mg2+ were in the same range, suggesting that Mg2+ ions mainly influence dissociation-association kinetics of the complex, with a minor contribution to its thermodynamic stability. Comparison of these two assays was shown to be useful in the analysis of thermodynamic and kinetic properties of protein:nucleic acid complexes

Plasmodium apicoplast tyrosyl-tRNA synthetase recognizes an unusual, simplified identity set in cognate tRNATyr

The life cycle of Plasmodium falciparum, the agent responsible for malaria, depends on both cytosolic and apicoplast translation fidelity. Apicoplast aminoacyl-tRNA synthetases (aaRS) are bacterial-like enzymes devoted to organellar tRNA aminoacylation. They are all encoded by the nuclear genome and are translocated into the apicoplast only after cytosolic biosynthesis. Apicoplast aaRSs contain numerous idiosyncratic sequence insertions: An understanding of the roles of these insertions has remained elusive and they hinder efforts to heterologously overexpress these proteins. Moreover, the A/T rich content of the Plasmodium genome leads to A/U rich apicoplast tRNA substrates that display structural plasticity. Here, we focus on the P. falciparum apicoplast tyrosyl-tRNA synthetase (Pf-apiTyrRS) and its cognate tRNATyr substrate (Pf-apitRNATyr). Cloning and expression strategies used to obtain an active and functional recombinant Pf-apiTyrRS are reported. Functional analyses established that only three weak identity elements in the apitRNATyr promote specific recognition by the cognate Pf-apiTyrRS and that positive identity elements usually found in the tRNATyr acceptor stem are excluded from this set. This finding brings to light an unusual behavior for a tRNATyr aminoacylation system and suggests that Pf-apiTyrRS uses primarily negative recognition elements to direct tyrosylation specificity.publishe

Accurate energies of hydrogen bonded nucleic acid base pairs and triplets in tRNA tertiary interactions

Tertiary interactions are crucial in maintaining the tRNA structure and functionality. We used a combined sequence analysis and quantum mechanics approach to calculate accurate energies of the most frequent tRNA tertiary base pairing interactions. Our analysis indicates that six out of the nine classical tertiary interactions are held in place mainly by H-bonds between the bases. In the remaining three cases other effects have to be considered. Tertiary base pairing interaction energies range from −8 to −38 kcal/mol in yeast tRNA(Phe) and are estimated to contribute roughly 25% of the overall tRNA base pairing interaction energy. Six analyzed posttranslational chemical modifications were shown to have minor effect on the geometry of the tertiary interactions. Modifications that introduce a positive charge strongly stabilize the corresponding tertiary interactions. Non-additive effects contribute to the stability of base triplets

Study of the structural requirements of E coli valine transfer RNA in aminoacylation and protein synthesis

The structural requirements for the function of the 3[superscript]\u27 CCA end of E. coli valine transfer RNA in aminoacylation and protein synthesis have been investigated, by studying the activities of 3[superscript]\u27 end tRNA[superscript] Val variants in these processes. These studies show that 3[superscript]\u27 end tRNA[superscript] Val variants can be obtained which are functional in both aminoacylation and later steps of protein synthesis. Replacement of the 3[superscript]\u27 terminal adenosine with either cytosine or uracil yields tRNAs that retain almost full aminoacylation activity (40-50% that of wild type tRNA[superscript] Val). The tRNA[superscript] Val variant with a 3[superscript]\u27-terminal guanine remains fully chargeable, but is a poor substrate for valyl-tRNA synthetase (VRS). Valyl-tRNA[superscript] Val with 3[superscript]\u27-CCG is active in in vitro poly(U,G)-directed (Val, Phe) copolypeptide synthesis, whereas valyl-tRNAs with 3[superscript]\u27-CCC or-CCU are inactive in this process. tRNA[superscript] Val variants at positions 74 and 75 can also be obtained which are functional in both aminoacylation and the later steps of protein synthesis;The determination of the identity elements of E. coli tRNA[superscript] Val has been investigated using two different approaches: first, to determine the sites on tRNA[superscript] Val that are recognized by VRS, aminoacylation kinetic studies of tRNA[superscript] Val variants, accompanied by [superscript]19F NMR spectrosopy of some tRN[superscript] Val variants, are carried out; second, to identify the already-known and additional recognition elements of tRNA[superscript] Val, and determine the negative determinants in noncognate tRNAs that prevent proper recognition of the tRNAs by VRS, the determination of valylation activities of tRNA[superscript] Ala and tRNA[superscript] Phe variants that contain tRNA[superscript] Val identity elements is carried out;These studies show that in addition to the anticodon bases A35 and C36, the discriminator base A73, and the base G20 in the variable pocket are probably recognized by VRS and are identity elements of tRNA[superscript] Val. In addition, the specific conformation of the acceptor stem around base pair U4-A69 may be important for the recognition of tRNA[superscript] Val by VRS. Furthermore, the upper part of the anticodon stem of tRNA [superscript] Val may also contain VRS recognition sites. Our studies suggest that the acceptor stem of E. coli tRNA [superscript] Ala, and the anticodon stem of E. coli tRNA[superscript] Phe contain negative recognition elements that prevent productive interactions between the tRNAs and VRS

Sequences outside recognition sets are not neutral for tRNA aminoacylation. Evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe.

Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363-1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia coli tRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a "classical" recognition element since its antideterminant effect is compensated for by a second-site mutation. This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name "permissive elements," are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently.journal articleresearch support, non-u.s. gov't1998 May 08importe

Mechanisms of leucyl-tRNA synthetase dependent group I intron splicing

Leucyl-tRNA synthetase (LeuRS) plays dual roles within the yeast mitochondria. In addition to protein synthesis, it is also essential to RNA splicing of critical respiratory genes. The LeuRS collaborates with a maturase to excise the bI4 and aI4α introns from the cob and cox1α genes respectively. The LeuRS-based suppressor mutations have been isolated within the amino acid editing CP1 domain and restore native RNA splicing activity in the presence of an inactive maturase. Mutational analysis of these sites and the regions that surround them demonstrated that certain substitutions can also inactivate LeuRS-dependent splicing activity under in vivo and in vitro conditions. Binding measurements suggest that these suppressor sites are important in maintaining interaction between LeuRS and the group I intron RNA. Thus, CP1 domain binds specifically to the bI4 and aI4α intron to promote RNA splicing. In addition to LeuRS from yeast mitochondria (ymLeuRS), diverse LeuRSs from varied origins such as M. tuberculosis and human mitochondria complement the ymLeuRS activities. Similarly, wild-type E. coli LeuRS (EcLeuRS) complemented a ymLeuRS null strain. Interestingly, at reduced levels of EcLeuRS expression in yeast mitochondria, the heterologous synthetase supported protein synthesis, but not intron splicing. Thus, it is a weak splicing suppressor. Surprisingly, a gain of splicing activity was exhibited by positive charge substitutions at the Ala293 position, suggesting that this Ala293 can be adapted for alternative activities. Preliminary footprinting data suggest that LeuRS binds to the P4-P6 core region of the bI4 intron that is cognate to LeuRS. The RNA duplex mimics of the P6 helix were designed and it was shown that LeuRS promotes their annealing in an ATP-independent manner. Domain analysis of LeuRS shows that the C-terminal domain is critical to the RNA annealing activity. Yeast mitochondrial tRNALeu (ymtRNALeu) competitively inhibit annealing. Also, an ymtRNALeu variable-stem-like region was identified on the P6 stem that is important for LeuRS-dependent annealing. These data support that the annealing and tRNA variable arm binding sites overlap on the C-terminal domain of LeuRS. It was shown that the overhang location and length of the duplexes are important features that LeuRS recognizes. It was hypothesized that LeuRS plays a key role in remodeling specific group I intron ribozymes so that they can productively self-splice