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PubMed1997

Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant.

The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.

Aspartate-tRNA Ligase↗

PubMedDate not supplied

Interaction of transfer RNA with 50S ribosomal subunits of Escherichia coli in absence of templates.

The equilibrium constant of a complex of tRNA with the 50S ribosomal subunit was measured in the absence of a template. It was shown that the stability of the complex increases with an increase in the concentration of Mg2+, it decreases with an increase in the concentration of univalent ions, and does not depend on the pH of the medium in the range of 7.0-8.2. Removal of the 3'-terminal nucleoside of tRNA weakens the association approximately 40-fold; the subsequent successive splitting off of another three nucleotides has little effect on the association constant. In 90% 2H2O the stability of the complex increases approximately four-fold, which points to the large contribution of the hydrogen bonds to the free energy of the interaction. The tetranucleotide TphiCG competes slightly with tRNA for sites on the 50S subparticles; this means that the TphiC segment of tRNA does not play an important role in the formation of the complex under investigation.

Base Sequence↗

PubMed2006

Crystal structure, conformation, and absolute configuration of kanamycin A.

Kanamycin, an antibiotic complex produced by Streptomyces kanamycetius isolated from Japanese soil, was described by Okami and Umezawa as early as 1957 and consists of three components: Kanamycin A (the major component), B, and C. The disulfate salt of kanamycin A [4-O-(6-amino-6-deoxy-alpha-d-glucopyranosyl)-6-O-(3-amino-3-deoxy-alpha-d-glucopyranosyl)-2-deoxystreptamine] is a broad-spectrum antibiotic that is used to treat gonorrhea, salmonella, tuberculosis, and many other diseases. Crystals of kanamycin A monosulfate monohydrate obtained from water are triclinic, space group P1, with a=7.2294(14), b=12.4922(15), c=7.1168(9), alpha=94.74(1), beta=89.16(1), gamma=91.59(1), V=640.2(2)A(3), micro(CuKalpha)=18.4cm(-1), FW 600.6, D(calc)=1.558g/cm(3), CAD-4 diffractometric data (2693 reflections, 25543sigma(I)), structure by shelx-86 and refined by full-matrix least squares to a final R value of 0.038. The wrong conformer had an R value of 0.043. Both of the d-glucose moieties are attached to the deoxystreptamine by alpha linkages. This absolute configuration agrees with the earlier determination by both chemical and X-ray methods with photographic data. The (phi,psi) values for the glycosidic linkages are 101.6 degrees , -121.1 degrees , 106.3 degrees , and -140.4 degrees , respectively. Kanamycin interacts with the ribosomal S12 protein to stabilize the codon-anticodon binding between mRNA and the aminoacyl tRNA and inhibits the elongation of peptide chains through a series of reactions resulting in the prevention of ribosomes from moving along mRNA.

Anti-Bacterial Agents↗

PubMed1991

Interaction of ribo- and deoxyriboanalogs of yeast tRNA(Phe) anticodon arm with programmed small ribosomal subunits of Escherichia coli and rabbit liver.

A synthetic ribooligonucleotide, r(CCAGACUGm-AAGAUCUGG), corresponding to the unmodified yeast tRNA(Phe) anticodon arm is shown to bind to poly(U) programmed small ribosomal subunits of both E. coli and rabbit liver with affinity two order less than that of a natural anticodon arm. Its deoxyriboanalogs d(CCAGACTGAAGATCTGG) and d(CCAGA)r(CUGm-AAGA)d(TCTGG), are used to study the influence of sugar-phosphate modification on the interaction of tRNA with programmed small ribosomal subunits. The deoxyribooligonucleotide is shown to adopt a hairpin structure. Nevertheless, as well as oligonucleotide with deoxyriboses in stem region, it is not able to bind to 30S or 40S ribosomal subunits in the presence of ribo-(poly(U] or deoxyribo-(poly (dT) template. The deoxyribooligonucleotide also has no inhibitory effect on tRNA(Phe) binding to 30S ribosomes at 10-fold excess over tRNA. Neomycin does not influence binding of tRNA anticodon arm analogs used. Complete tRNA molecule and natural modifications of anticodon arm are considered to stabilize the arm structure needed for its interaction with a programmed ribosome.

Animals↗

PubMed2003

RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis.

Vigilin/Scp160p/DDP1 is a ubiquitous and highly conserved protein containing 15 related, but non-identical, K-homology (KH) nucleic acid binding domains. While its precise function remains unknown, proposed roles for vigilin include chromosome partitioning at mitosis, facilitating translation and tRNA transport, and control of mRNA metabolism, including estrogen-mediated stabilization of vitellogenin mRNA. To probe sites of vigilin action in vertebrate cells, we performed nucleic acid binding and RNA interference studies. Consistent with a potential role in chromosome partitioning, human vigilin exhibits a higher affinity for Drosophila dodecasatellite single-stranded DNA than for vitellogenin mRNA 3'-UTR. Direct observation and flow cytometry in non-mitotic, serum-starved, HeLa cells showed that RNAi-mediated vigilin knockdown is rapidly lethal, indicating an essential function for vigilin distinct from its proposed role in chromosome partitioning. Pulse labeling experiments revealed that rates of protein synthesis and degradation are unaffected by the several fold reduction in vigilin levels early in siRNA knockdown indicating that vigilin is not a global regulator of translation. These data show that vigilin is an essential protein in human cells, support the view that vigilin's most essential functions are neither chromosome partitioning nor control of translation, and are consistent with vigilin playing a critical role in cytoplasmic mRNA metabolism.

3' Untranslated Regions↗

PubMed2004

Interdomain communication between weak structural elements within a disease-related human tRNA.

The structure of the human mitochondrial (hs mt) tRNALeu(UUR) features several domains that are predicted to exhibit limited thermodynamic stability. An elevated frequency of disease-related mutations within these domains suggests a link between structural instability and the functional effects of pathogenic mutations. A series of tRNAs featuring mutations within the D and anticodon stems were prepared and investigated using nuclease probing. Structural mapping studies indicated that these domains were partially denatured for the wild type (WT) hs mt tRNALeu(UUR) and were significantly stabilized by mutations introducing additional or stronger base pairs into the stem regions. In addition, trends in the aminoacylation activities of the D stem mutants suggested that the loose structure is required for function, with mutants displaying the most ordered structures exhibiting the lowest levels of aminoacylation activity. A pronounced interdependence of the structures of the anticodon and D stems was observed, with mutations strengthening the D stem stabilizing the anticodon stem and vice versa. The existence of strong interdomain communication was further elucidated with a mutant of hs mt tRNALeu(UUR) containing a stabilized D stem and a pathogenic mutation that disrupted the anticodon stem. Strengthening the structure of the D stem completely restored the function of the disease-related mutant to WT levels, indicating that propagated structural weaknesses contribute to the functional deactivation of this tRNA by mutations.

Acylation↗

PubMed2002

Novel complexes of mammalian translation elongation factor eEF1A.GDP with uncharged tRNA and aminoacyl-tRNA synthetase. Implications for tRNA channeling.

Multimolecular complexes involving the eukaryotic elongation factor 1A (eEF1A) have been suggested to play an important role in the channeling (vectorial transfer) of tRNA during protein synthesis [Negrutskii, B.S. & El'skaya, A.V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47-78]. Recently we have demonstrated that besides performing its canonical function of forming a ternary complex with GTP and aminoacyl-tRNA, the mammalian eEF1A can produce a noncanonical ternary complex with GDP and uncharged tRNA [Petrushenko, Z.M., Negrutskii, B.S., Ladokhin, A.S., Budkevich, T.V., Shalak, V.F. & El'skaya, A.V. (1997) FEBS Lett. 407, 13-17]. The [eEF1A.GDP.tRNA] complex has been hypothesized to interact with aminoacyl-tRNA synthetase (ARS) resulting in a quaternary complex where uncharged tRNA is transferred to the enzyme for aminoacylation. Here we present the data on association of the [eEF1A.GDP.tRNA] complex with phenylalanyl-tRNA synthetase (PheRS), e.g. the formation of the above quaternary complex detected by the gel-retardation and surface plasmon resonance techniques. To estimate the stability of the novel ternary and quaternary complexes of eEF1A the fluorescence method and BIAcore analysis were used. The dissociation constants for the [eEF1A.GDP.tRNA] and [eEF1A.GDP.tRNAPhe.PheRS] complexes were found to be 20 nm and 9 nm, respectively. We also revealed a direct interaction of PheRS with eEF1A in the absence of tRNAPhe (Kd = 21 nm). However, the addition of tRNAPhe accelerated eEF1A.GDP binding to the enzyme. A possible role of these stable novel ternary and quaternary complexes of eEF1A.GDP with tRNA and ARS in the channeled elongation cycle is discussed.

Amino Acyl-tRNA Synthetases↗

PubMed2000

Hypermodified nucleosides in the anticodon of tRNALys stabilize a canonical U-turn structure.

Modified nucleosides in the anticodon domain of Escherichia coli tRNA(Lys) are necessary for high-affinity codon recognition and reading frame maintenance. Human tRNA(Lys,3) is the specific primer for HIV-1 reverse transcriptase and also requires nucleoside modification for proper function. We now present NMR solution structures for the fully modified 17-nucleotide E. coli tRNA(Lys) anticodon stem-loop domain (ASL). NMR data were also collected for several partially modified ASLs, revealing the contributions each modified nucleoside (mnm(5)s(2)U34, t(6)A37, and psi39) makes in transforming the disordered, unmodified tRNA ASL into the highly ordered native structure. The solution structure of the native ASL domain provides insight into longstanding questions regarding both wobble position modification and the nearly ubiquitous t(6)A37 found in tRNAs with an adjacent U at position 36. Native tRNA(Lys) has a U-turn structure similar to the yeast tRNA(Phe) crystal structure, unlike previously proposed "unconventional" anticodon structures characterized by stable interactions between mnm(5)s(2)U-34 and t(6)A-37.

Anticodon↗

PubMed1998

Abnormal proteins enhance stress-induced cell death.

The effect of abnormal proteins on cell viability was studied using artificially cleaved polypeptides. Escherichia coli methionyl-tRNA synthetase (MetRS) consists of two distinct domains and its activity is essential for cell viability. The polypeptide chain was split by linker insertion and expressed as two fragments. Two pairs of polypeptides, one split within the N-terminal domain and another at the junction of the two domains retained aminoacylation activity. The in vitro activities of these split mutants were enhanced by the presence of chaperonin, GroESL. However, cells containing these split polypeptides became sensitive to conditions that induce GroESL. The results of this work suggest that an abnormally generated protein can cause cell death under stressful conditions.

Anti-Infective Agents↗

PubMed2005

Characterizing the function and structural organization of the 5' tRNA-like motif within the hepatitis C virus quasispecies.

Hepatitis C virus (HCV) RNA is recognized and cleaved in vitro by RNase P enzyme near the AUG start codon. Because RNase P identifies transfer RNA (tRNA) precursors, it has been proposed that HCV RNA adopts structural similarities to tRNA. Here, we present experimental evidence of RNase P sensitivity conservation in natural RNA variant sequences, including a mutant sequence (A368-G) selected in vitro because it presented changes in the RNA structure of the relevant motif. The variation did not abrogate the original RNase P cleavage, but instead, it allowed a second cleavage at least 10 times more efficient, 4 nt downstream from the original one. The minimal RNA fragment that confers sensitivity to human RNase P enzyme was located between positions 299 and 408 (110 nt). Therefore, most of the tRNA-like domain resides within the viral internal ribosome entry site (IRES) element. In the variant, in which the mutation stabilizes a 4 nt stem-loop, the second cleavage required a shorter (60 nt) substrate, internal to the minimal fragment substrate, conforming a second tRNA-like structure with similarities to a 'Russian-doll' toy. This new structure did not impair IRES activity, albeit slightly reduced the efficiency of translation both in vitro and in transfected cells. Conservation of the original tRNA-like conformation together with preservation of IRES activity points to an essential role for this motif. This conservation is compatible with the presence of RNA structures with different complexity around the AUG start codon within a single viral population (quasispecies).

5' Untranslated Regions↗

PubMed1997

RNA splicing ligase activity in the archaeon Haloferax volcanii.

At least two separate enzymes, an endonuclease and a ligase, appear to be involved in tRNA splicing in halophilic archaea. We have identified and partially characterized a splicing ligase activity in cell extracts of Haloferax volcanii that can ligate deproteinized exon products generated in a separate endonuclease reaction. As in vitro transcribed partial intron-deleted derivative of H. volcanii elongator tRNA(Met) is used as substrate for the endonuclease. The ligase can also join the two exons that are independently eluted from the gels. This ligase activity is observed at a range (50 mM to 2.8 M) of monovalent cations in the assays, but is abolished when the enzyme preparations are depleted of the monovalent cations. In contrast, H. volcanii splicing endonuclease has been reported to require divalent cations and is inhibited by monovalent cations. Our endonuclease assays confirm these reports, and also show that the endonuclease is not permanently inactivated even in high monovalent cation containing extracts. The ligase activity in the extracts does not appear to require any divalent cation or exogenously added source of energy or phosphate.

Archaea↗

PubMed2002

tRNA-mediated transcription antitermination in vitro: codon-anticodon pairing independent of the ribosome.

Uncharged tRNA acts as the effector for transcription antitermination of genes in the T box family in Bacillus subtilis and other Gram-positive bacteria. Genetic studies suggested that expression of these genes is induced by stabilization of an antiterminator element in the leader RNA of each target gene by the cognate uncharged tRNA. The specificity of the tRNA response is dependent on a single codon in the leader, which was postulated to pair with the anticodon of the corresponding tRNA. It was not known whether the leader RNA-tRNA interaction requires additional factors. We show here that tRNA-dependent antitermination occurs in vitro in a purified transcription system, in the absence of ribosomes or accessory factors, demonstrating that the RNA-RNA interaction is sufficient to control gene expression by antitermination. The tRNA response exhibits similar specificity in vivo and in vitro, and the antitermination reaction in vitro is independent of NusA and functions with either B. subtilis or Escherichia coli RNA polymerase.

Anticodon↗

PubMed1991

Catalytic activity of aminoacyl tRNA synthetases and its implications for the origin of life. I. Aminoacyl adenylate formation in tyrosyl tRNA synthetase.

The changes in the catalytic activity resulting from amino acid substitutions in the active site region have been theoretically modeled for tyrosyl tRNA synthetase (Tyr-RS). The catalytic activity was calculated as the differential stabilization of the transition state using electrostatic approximation. The results indicate that charged residues His45, His48, Asp78, Asp176, Asp194, Lys225, Lys230, Lys233, Arg265, and Lys268 play essential roles in catalysis of aminoacyl adenylate formation in Tyr-RS, which is in general agreement with previously known experimental data for residues 45, 48, 194, 230, and 233. These catalytic residues have also been used to search for sequence homology patterns among class I aminoacyl RSs of which HIGH and KMSKS conserved sequence motifs are well known. His45 and His48 belong to the HIGH signature sequence of class I aminoacyl tRNA synthetases (aRSs), whereas Arg265 and Lys268 can constitute a part of the KMSKS charge pattern. Lys225, Lys230, and Lys233 may be part of the conservative substitution pattern [HKR]-X(4)-[HKR]-X(2)-[HKR], and Asp194 is part of the new GSDQ motif. This demonstrates that the three dimensional charge distribution near the active site is an essential feature of the catalytic activity of aRS and that the theoretical technique used in this work can be utilized in searches for the catalytically important residues that may provide a clue for a charge residue pattern conserved in evolution. The appearance of patterns I-IV in Arg-, Gln-, Met-, Ile-, Leu-, Trp-, Val-, Glu-, Cys-, and Tyr-RS indicates that all these enzymes could have the same ancestor.

Amino Acid Sequence↗

PubMed2000

Crucial role of the high-loop lysine for the catalytic activity of arginyl-tRNA synthetase.

The presence of two short signature sequence motifs (His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys (KMSK)) is a characteristic of the class I aminoacyl-tRNA synthetases. These motifs constitute a portion of the catalytic site in three dimensions and play an important role in catalysis. In particular, the second lysine of the KMSK motif (K2) is the crucial catalytic residue for stabilization of the transition state of the amino acid activation reaction (aminoacyl-adenylate formation). Arginyl-tRNA synthetase (ArgRS) is unique among all of the class I enyzmes, as the majority of ArgRS species lack canonical KMSK sequences. Thus, the mechanism by which this group of ArgRSs achieves the catalytic reaction is not well understood. Using three-dimensional modeling in combination with sequence analysis and site-directed mutagenesis, we found a conserved lysine in the KMSK-lacking ArgRSs upstream of the HIGH sequence motif, which is likely to be a functional counterpart of the canonical class I K2 lysine. The results suggest a plausible partition of the ArgRSs into two major groups, on the basis of the conservation of the HIGH lysine.

Amino Acid Sequence↗

PubMed1992

Assignment of the magnetic resonances of the imino protons and methyl protons of Bombyx mori tRNA(GlyGCC) and the effect of ion binding on its structure.

The magnetic resonances in the low-field H-NMR spectra of Bombyx mori tRNA(GlyGCC), corresponding to the hydrogen-bonded imino protons of the helical stems and tertiary base pairs, could be tentatively assigned by means of the sequential nuclear Overhauser effects. While B. mori tRNA(GlyGCC) does not contain the G19C56 tertiary base pair, the D20G57 base pair exists between the D and T loops, which was not found in the X-ray crystal structure of yeast tRNA(Phe). The effects of Mg2+, spermine and temperature on the conformation of this tRNA have also been examined based on the behavior of the assigned resonance signals. Mg2+ stabilize the D and T stems and the tertiary structure between the D and T loops. Spermine affects the resonances of the D and anticodon stems, and A23G9, but does not stabilize them. While the acceptor stem melts sequentially from both ends (G7C66 and G1C72) with increasing temperature, the anticodon stem melts from only one end (G39C31) and the G26C44 base pair is the most stable. In the tertiary structure between the variable loop and D stem, G10G45 melts first and G22G46 last. Yeast tRNA(Phe) has also been examined, and the results were compared with those for B. mori tRNA(Gly).

Animals↗

PubMed2001

Increased bar minigene mRNA stability during cell growth inhibition.

Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl-tRNA hydrolase (Pth) activity. Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth-defective cells. Two of these wild-type bar regions, barI+ and barII+, contain minigenes with similar AUG-AUA-stop codon sequences preceded by different Shine-Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two-codon 'ORF' (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two-codon 'ORF' sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA.

Bacterial Proteins↗

PubMed2001

The relationship between translational control and mRNA degradation for the Escherichia coli threonyl-tRNA synthetase gene.

Expression of thrS, the gene encoding Escherichia coli threonyl-tRNA synthetase, is negatively autoregulated at the translational level. Regulation is due to the binding of threonyl-tRNA synthetase to its own mRNA at a site called the operator, located immediately upstream of the initiation codon. The present work investigates the relationship between regulation and mRNA degradation. We show that two regulatory mutations, which increase thrS expression, cause an increase in the steady-state mRNA concentration. Unexpectedly, however, the half-life of thrS mRNA in the derepressed mutants is equal to that of the wild-type, indicating that mRNA stability is independent of the repression level. All our results can be explained if one assumes that thrS mRNA is either fully translated or immediately degraded. The immediately degraded RNAs are never detected due to their extremely short half-lives, while the fully translated messengers share the same half-lives, irrespective of the mutations. The increase in the steady-state level of thrS mRNA in the derepressed mutants is simply explained by an increase in the population of translated molecules, i.e. those never bound by the repressor, ThrRS. Despite this peculiarity, thrS mRNA degradation seems to follow the classical degradation pathway. Its stability is increased in a strain defective for RNase E, indicating that an endonucleolytic cleavage by this enzyme is the rate-limiting process in degradation. We also observe an accumulation of small fragments corresponding to the 5' end of the message in a strain defective for polynucleotide phosphorylase, indicating that, following the endonucleolytic cleavages, fragments are normally degraded by 3' to 5' exonucleolytic trimming. Although mRNA degradation was suspected to increase the efficiency of translational control based on several considerations, our results indicate that inhibition of mRNA degradation has no effect on the level of repression by ThrRS.

Endoribonucleases↗

PubMed1993

Evidence for distinct locations for metal binding sites in two closely related class I tRNA synthetases.

Of the ten class I tRNA synthetases, those for methionine and isoleucine are among the most closely related. In recent work we showed that the 676 amino acid E. coli methionine tRNA synthetase has one zinc bound per polypeptide. Zinc may be replaced by spectroscopically observable cobalt with retention of full activity. Bound zinc has been localized to a cysteine cluster within an insertion into the nucleotide binding fold that characterizes all class I enzymes. Mutations which interfere with metal ligation to these cysteines yield proteins that are defective in activity. Additional data presented here show that change of the cobalt oxidation state and coordination geometry of the Co(II)-substituted enzyme results in a complete loss in activity, and that mutations which replace any one of the zinc-binding cysteine sulfhydryls have a small but measurable effect on protein stability. These results further support the importance of the metal for the active site. We also show that, in contrast to methionine tRNA synthetase, the closely related but larger 939 amino acid E. coli isoleucine tRNA synthetase contains 1.5 to 2 molecules of zinc bound per polypeptide. The cobalt-substituted enzyme is active and shows the expected spectrum for tetrahedral coordination to sulfur ligands. Although the site(s) for metal coordination in isoleucine tRNA synthetase has not been rigorously established, one likely sequence element is in a region of the primary structure different from the known metal binding site in methionine tRNA synthetase. Thus, these two closely related proteins have incorporated metal binding sites into distinct parts of their related sequences.

Amino Acid Sequence↗