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PubMed1977

Effect of magnesium and polyamines on the structure of yeast tRNAPhe.

The effect of magnesium and polyamines (spermine, spermidine, putrescine and cadaverine) on the structure of yeast tRNAPhe has been investigated. It is found that magnesium induces structural changes and stabilizes hydrogen bonds in the temperature range 22--44 degrees C in 0.17 M sodium. The number of Mg2+ which affect tRNA structure increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C and the number of additional base pairs formed in the presence of magnesium increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C. The spectral changes are more-or-less sequential. The polyamine spermine stabilizes some, but not all, of the structural features stabilized by magnesium at 44 degrees C, and the combination of magnesium and spermine, at low levels, is more effective than either cation alone in stabilizing tRNA structure. Comparison of the effects of spermine, spermidine, putrescine and cadaverine indicates that it is the asymmetric triamine unit which is important in the stabilization. Some spectral changes induced by magnesium can be assigned to stabilization of specific tertiary structure interactions and to alteration of stacking adjacent to U8-A14.

Magnesium↗
PubMed1972

Hydrostatic pressure effects on protein synthesis.

The effects of high hydrostatic pressure on several phases of cell-free protein synthesis have been examined. The initial rate of polyuridylic acid (poly U)-directed synthesis of polyphenylalanine showed an apparent increase at 100 atm, above which the synthetic rate was reduced sharply with increased pressure up to 640 atm where 95% inhibition was observed. The magnitude of the inhibition of polyphenylalanine synthesis with increased pressure depended strongly on the magnesium salt concentration in the reaction system. Misreading of the poly U message, as measured by insertion of leucine in place of phenylalanine, dropped rapidly with increased pressure from 1 to 350 atm, above which the amount of misreading increased. Enzymatic activation of transfer RNAs (tRNAs) was reduced by increased pressure in the range 100-640 atm, where the rate of tRNA aminoacylation was 80% inhibited. Both nonenzymatic attachment of phenylalanyl-tRNA (phe-tRNA) to the poly U-ribosome complex and stability of the phe-tRNA-poly U-ribosome complex were decreased at high pressures (100-900 atm). The results of the action of pressure on the various phases of cell-free protein synthesis suggest that the major pressure-sensitive element in the protein synthetic machinery is the ribosome.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Interaction of mRNA analogs--oligoribonucleotides AUGU3, AUGU6 and (pU)6 with 80S ribosomes from human placenta in the presence of related tRNA and a protein-synthesizing system].

Binding of oligoribonucleotides AUGUn (n-3 and 6) and (pU) to 80S ribosomes from human placenta in the presence of cognate tRNAs and a "ribosome-free" protein-synthesizing system from rabbit reticulocytes has been studied. The binding of the mRNA analogues resulted in formation of stable post-translocational complexes (which may be easily isolated by centrifugation in sucrose density gradient): 80S.AUGU3.MetPhe-tRNA(Phe); 80S.AUGU6.Met(Phe)2.tRNA(Phe); 80S.(pU)6.(Phe)2-tRNA(Phe). In these complexes the ratios of the bound ligands are close to the theoretically expected values. Comparison of the results obtained with the previously reported data on nonenzymatic binding of oligouridylates and Phe-tRNA(Phe) to 80S ribosomes lead one to the conclusion that translation factors significantly stabilize the complexes of tRNA with 80S ribosomes and oligoribonucleotide templates.

Binding Sites↗
PubMed1994

Effector region of the translation elongation factor EF-Tu.GTP complex stabilizes an orthoester acid intermediate structure of aminoacyl-tRNA in a ternary complex.

tRNA(Val) from Escherichia coli was aminoacylated with [1-13C]valine and its complex with Thermus thermophilus elongation factor EF-Tu.GTP was analyzed by 13C NMR spectroscopy. The results suggest that the aminoacyl residue of the valyl-tRNA in ternary complex with bacterial EF-Tu and GTP is not attached to tRNA by a regular ester bond to either a 2'- or 3'-hydroxyl group; instead, an intermediate orthoester acid structure with covalent linkage to both vicinal hydroxyls of the terminal adenosine-76 is formed. Mutation of arginine-59 located in the effector region of EF-Tu, a conserved residue in protein elongation factors and the alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), abolishes the stabilization of the orthoester acid structure of aminoacyl-tRNA.

Binding Sites↗
PubMed1993

5-Methylcytidine is required for cooperative binding of Mg2+ and a conformational transition at the anticodon stem-loop of yeast phenylalanine tRNA.

The role of modified nucleosides in tRNA structure and ion binding has been investigated with chemically synthesized RNAs corresponding to the yeast tRNA(Phe) anticodon stem and loop (tRNA(ACPhe). Incorporation of d(m5C) at position 14 of the stem of tRNA(ACPhe)-d(m5C14), CCAGACUGAAGAU-d(m5C14)-UGG, analogous to m5C40 in native tRNA(Phe), introduced a strong Mg2+ binding at a site distant from the m5C. A Mg(2+)-induced structural transition, detected by circular dichroism spectroscopy, was similar to that observed for the DNA analog of tRNA(ACPhe) (Guenther et al., 1992; Dao et al., 1992). In contrast, Mg2+ had little effect on unmodified tRNA(ACPhe)-rC14 or tRNA(ACPhe)-d(C14). Modified tRNA(ACPhe)-d(m5C14) bound two Mg2+ ions, and the binding was cooperative. The dissociation constant of the two Mg2+ ions from tRNA(ACPhe)-d(m5C14), 2.5 x 10(-9) M2, is the result of an RNA structure significantly stabilized by Mg2+ binding, delta G = -11.7 kcal/mol. The tRNA(ACPhe)-d(m5C14) structure, investigated by 1H NMR, had a double stranded stem of five base pairs and two additional base pairs across what was a seven membered loop in the unmodified tRNA(Phe)AC. Methylation of cytidine in the yeast tRNA(ACPhe) enables the molecule to form more than one conformation through a process regulated by Mg2+ concentration. Thus, the simplest of posttranscriptional modifications of tRNA, a methylation, is involved in a somewhat distant, internal-site Mg2+ binding and stabilization of tRNA structure, especially that of the anticodon stem and loop.

Anticodon↗
PubMed2000

Energetic contribution of tRNA hybrid state formation to translocation catalysis on the ribosome.

Upon transpeptidylation, the 3' end of aminoacyl-tRNA (aa-tRNA) in the ribosomal A site enters the A/P hybrid state. We report that transpeptidylation of Phe-tRNA to fMetPhe-tRNA on Escherichia coli ribosomes substantially lowers the kinetic stability of the ribosome-tRNA complex and decreases the affinity by 18.9 kJ mol(-1). At the same time, the free energy of activation of elongation factor G dependent translocation decreases by 12.5 kJ mol(-1), indicating that part of the free energy of transpeptidylation is used to drive translocation kinetically. Thus, the formation of the A/P hybrid state constitutes an important element of the translocation mechanism.

Binding Sites↗
PubMed2004

Monitoring uncharged tRNA during transcription of the Bacillus subtilis glyQS gene.

Expression of the Bacillus subtilis glyQS gene, encoding glycyl-tRNA synthetase, depends on stabilization of an antiterminator element during transcription of the 5' region of the mRNA by binding of uncharged tRNA(Gly). The glyQS gene is a member of the T box family of genes, all of which are involved in generation of charged tRNA. Each gene in this family exhibits an increase in readthrough of a termination signal located upstream of the start of the coding sequence in response to a decrease in the ratio of charged to uncharged tRNA. Many structural features of T box RNAs that are necessary for tRNA-dependent antitermination have been defined, but little is known about the timing or sequence of events that lead to a productive interaction with uncharged tRNA and discrimination against charged tRNA. To investigate these issues, transcription complexes were blocked artificially at specific positions along the leader sequence and tested for the ability to recognize tRNA. Although the sequence element that binds the tRNA anticodon is located more than 100 nt before the termination signal, complexes with nascent transcripts extending to just upstream of the termination site were still competent for antitermination. This result indicates that the transcript can fold into a receptive structure in the absence of the tRNA, and that tRNA is not necessary prior to this point. A mimic of charged tRNA(Gly) inhibited antitermination by uncharged tRNA unless the leader RNA-tRNA(Gly) complexes contained the complete antiterminator. These results suggest that the transcription complex can interact with either uncharged or charged tRNA until it approaches the termination point, allowing maximal flexibility in monitoring the ratio of charged to uncharged tRNA.

Bacillus subtilis↗
PubMed1991

Structural analyses on yeast tRNA(Tyr) and its complex with tyrosyl-tRNA synthetase by the use of hydroxyl radical 'footprinting'.

Hydroxyl radical, generated by reduction of hydrogen peroxide by Fe(II)-EDTA, was used to investigate the contact sites of yeast tRNA(Tyr) with its cognate tyrosyl-tRNA synthetase (TyrRS). Exposure of free tRNA(Tyr) to this reagent gave cleavage patterns consistent with the tertiary structure of yeast tRNA(Phe) established by X-ray crystallography. When the probing reaction was performed under the conditions which stabilized complex formation between tRNA(Tyr) and TyrRS, aminoacyl-stem region of the tRNA was protected from cleavage. This result supports our earlier finding that the information for binding to TyrRS would reside mainly in the aminoacyl-stem of tRNA(Tyr).

Binding Sites↗
PubMedDate not supplied

[Differences in the thermostability of aminoacyl-mRNA-synthetases from intact and denervated rabbit muscles].

The stability of alanyl-tRNA synthetase and valyl-tRNA synthetase from the intact and denervated for 30 days rabbit skeletal muscles towards the inactivating effect of heat (42 degrees for 10 min) has been studied. The activity of the enzymes was measured in the supernatant fraction obtained by centrifugation of muscle homogenates at 105, 000 g for 1 h. The stability of both alanyl- and valyl-tRNA synthetases from the denervated m. soleus and m. gastrocnemius was shown to be considerably decreased as compared with the enzymes from the intact muscles.

Alanine-tRNA Ligase↗
PubMed1989

Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme.

MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA aminoacylation step than on the adenylate formation step. The Km for ATP and methionine are unaltered indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3 which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the three-dimension structure of the homologous E. coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide-binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration to the catalytic residues.

Amino Acid Sequence↗
PubMed1995

Permutation of a pair of tertiary nucleotides in a transfer RNA.

The tertiary nucleotides at positions 15 and 48 in a tRNA establish non-Watson-Crick hydrogen interactions that connect the dihydrouridine (D) loop with the variable loop and stabilize the "L"-shaped tRNA structure. Although the majority of tRNAs have G15.C48 or A15.U48, all of the 16 possible nucleotide pairs at positions 15 and 48 can be found in the existing cytoplasmic and mitochondrial tRNA sequences. Because tRNAs contain a variety of slightly different sets of tertiary nucleotides, this complexity raises the question of whether a given tRNA sequence framework can accommodate all of the 16 compositions at positions 15 and 48. In this work, G15 and C48 in an Escherichia coli alanine amber suppressor tRNA were permuted, and variants were tested for biological activity in vivo. All but an A15.A48 variant were functional, indicating substantial flexibility at positions 15 and 48 to accommodate nucleotide variations. Analysis of the A15.A48 variant with chemical probes showed that this mutant harbors a defect that specifically changes the conformation of the anticodon sequence. Interestingly, human tRNA(Ala) has A15.A48. Additional nucleotide substitutions in E. coli A15.A48 tRNA(Ala) that recreate the D loop sequence of human tRNA(Ala) restored the biological activity to this tRNA by reestablishing the wild-type conformation of the anticodon sequence. The results suggest a distal relationship between the D and the anticodon loops in a tRNA and delineate covariation of specific nucleotides in the evolution of tRNA(Ala) from E. coli to human.

Anticodon↗
PubMed1982

Studies on the high molecular weight form of polypeptide chain elongation factor-1 from pig liver. II. Interaction with guanine nucleotides and aminoacyl-tRNA.

The high molecular weight form of polypeptide chain elongation factor-1, or EF-1H, has been purified from pig liver to apparent homogeneity and shown to be a 1:1:1 complex of EF-1 alpha, EF-1 beta, and EF-1 gamma, namely, EF-1 alpha beta gamma (Hattori, S. and Iwasaki, K. (1980) J. Biochem. 88, 725-736). Therefore, its enzymatic properties were investigated in detail. The effects of various components such as NH4Cl, Mg(CH3COO)2, guanine nucleotides and Phe-tRNA on its heat stability were investigated and it was found that its properties were very close to those of the aminoacyl-tRNA binding enzyme from rabbit reticulocytes (McKeehan, W.L. and Hardesty, B. (1969) J. Biol. Chem. 244, 4330-4339). Furthermore, it was reported that EF-1 alpha beta gamma seemed to dissociate into EF-1 alpha and EF-1 beta gamma when guanine nucleotides were present, and also to form a complex of EF-1 alpha . GTP . [14C]Phe-tRNA when both GTP and [14C]Phe-tRNA were present. On the contrary, however, when the interaction between EF-1 alpha beta gamma and guanine nucleotides was analyzed by gel filtration at 4 degrees C, the dissociation of EF-1 alpha beta gamma could not be detected, instead the formation of binary complexes containing EF-1 alpha beta gamma and guanine nucleotides was observed. The dissociation constants of the EF-1 alpha beta gamma . GDP and EF-1 alpha beta gamma . GTP complexes were estimated to be 6.8 x 10(-6) M and 7.3 x 10(-6) M, respectively, and the number of binding sites per molecule of EF-1 alpha beta gamma for these nucleotides was estimated to be one. Little, if any, interaction was detected between EF-1 alpha beta gamma and aminoacyl-tRNA even in the presence of GTP. The controversial results obtained by different methods as described above seemed to be compatible with each other, if one assumes that the dissociation constant for the conversion of EF-1 alpha beta gamma into EF-1 alpha and EF-1 beta gamma increased with the increase in temperature. When gel filtration of EF-1 alpha beta gamma was carried out with a solution containing both GTP and [14C]Phe-tRNA, the formation of a ternary complex containing EF-1 alpha, GTP and [14C]Phe-tRNA was detected at 4 degrees C, although the amount was very small. From these results, we assumed that EF-1 alpha beta gamma interacts with GTP and aminoacyl-tRNA (aa-tRNA) according to the following sequences; EF-1 alpha beta gamma + GTP in equilibrium with EF-1 alpha beta gamma . GTP EF-1 alpha beta gamma . GTP in equilibrium with EF-1 alpha . GTP + EF-1 beta gamma EF-1 alpha . GTP + aa-tRNA in equilibrium with EF-1 alpha . GTP . aa-tRNA.

Animals↗
PubMed1995

Recognition of tRNAPhe by phenylalanyl-tRNA synthetase of Thermus thermophilus.

The tRNA(Phe) nucleotides required for recognition by phenylalanyl-tRNA synthetase of Thermus thermophilus have been determined using Escherichia coli tRNA(Phe) transcripts with various mutations. The anticodon nucleotides are shown to be the most important recognition elements. The discriminator nucleotide, A73, involved in the recognition set of yeast, E. coli and human phenylalanyl-tRNA synthetases contributes only slightly to tRNA(Phe) recognition by Th. thermophilus phenylalanyl-tRNA synthetase. Nucleotide 20 and some tertiary nucleotides, including the conserved G19.C56 base pair, are proposed to participate in stabilization of the precise tRNA conformation required for efficient aminoacylation. The role of the 3'-CCA terminus, common to all tRNAs, in the specific interaction of tRNA with phenylalanyl-tRNA synthetase is discussed.

Anticodon↗
PubMed1992

Aminoacyl-tRNA synthetase-induced cleavage of tRNA.

Aminoacyl-tRNA synthetases interact with their cognate tRNAs in a highly specific fashion. We have examined the phenomenon that upon complex formation E. coli glutaminyl-tRNA synthetase destabilizes tRNA(Gln) causing chain scissions in the presence of Mg2+ ions. The phosphodiester bond cleavage produces 3'-phosphate and 5'-hydroxyl ends. This kind of experiment is useful for detecting conformational changes in tRNA. Our results show that the cleavage is synthetase-specific, that mutant and wild-type tRNA(Gln) species can assume a different conformation, and that modified nucleosides in tRNA enhance the structural stability of the molecule.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Mutational analysis of the joining regions flanking helix P18 in E. coli RNase P RNA.

We have studied variants of Escherichia coli RNase P RNA with base exchanges in the joining regions flanking helix P18, which form part of the ribozyme core structure. Mutant RNase P RNAs were analyzed for: (1) specific tRNA binding by gel retardation; (2) catalytic performance in single turnover reactions; (3) structural perturbations utilizing Pb2+ -induced hydrolysis; and (4) in vivo function by complementation analysis in E. coli RNase P mutant strains. Our in vitro experiments revealed that the base moieties of nucleotides (nt) 303 and 331 to 333 neither significantly contribute to tRNA binding or structural stabilization of RNase P RNA nor to active site chemistry. Single base exchanges at nt 300, 301, and 330 reduced tRNA binding, while having little effect on the catalytic rate, which demonstrates that these nucleotides are involved in forming the high affinity (pre-)tRNA binding site. In contrast, point mutations at the strictly conserved positions nt 328, 329, 334 and 335 reduced tRNA binding affinity as well as the catalytic rate, suggesting that these mutations additionally disrupted important interactions in the catalytic center. Probing by Pb2+ revealed that particularly the mutations that affected catalytic function most strongly perturbed a more extended region (nt 248 to 335) known to be involved in tRNA binding. Under high salt conditions (> or = 0.8 M NH4+), catalytic defects of the mutant RNase P RNAs were much less pronounced, suggesting that structural perturbations leading to increased electrostatic repulsion between phosphate groups were the main cause for observed functional defects. Only mutant C334 retained a largely increased pre-steady-state K(m(pss)) under high salt conditions. We conclude that the base at position 334 is directly involved in a contact crucial to pre-tRNA binding. A complementation analysis demonstrated the important role in vivo of the joining regions flanking helix P18. None of the bases could be mutated without affecting bacterial viability.

Base Composition↗
PubMed1999

Structure-specific tRNA-binding protein from the extreme thermophile Aquifex aeolicus.

The genome of the bacterium Aquifex aeolicus encodes a polypeptide which is related to a small portion of a sequence found in one prokaryotic and two eukaryotic tRNA synthetases. It also is related to a portion of Arc1p, a tRNA-binding protein believed to be important for nuclear trafficking of tRNAs. Here we cloned, expressed and purified the 111 amino acid polypeptide (designated Trbp111) and showed by ultracentrifugation analysis that it is a stable dimer in solution. The protein was also crystallized in a monoclinic lattice. X-ray diffraction analysis at 2.8 A resolution revealed a prominent non-crystallographic 2-fold axis, consistent with the presence of a symmetric homodimeric structure. Band-shift analysis with polyacrylamide gels showed that the dimer binds tRNAs, but not RNA duplexes, RNA hairpins, single-stranded RNA nor 5S rRNA. Complex formation with respect to tRNA is non-specific, with a single tRNA bound per dimer. Thus, Trbp111 is a structure-specific tRNA-binding protein. These results and other considerations raise the possibility that Trbp111 is a tRNA-specific chaperone which stabilizes the native L-shaped fold in the extreme thermophile and which has been incorporated into much larger tRNA-binding proteins of higher organisms.

Amino Acid Sequence↗
PubMed2005

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine.

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.

Crystallography, X-Ray↗