Chemical and physical studies on the structure of the histidyl transfer ribonucleic acid synthetase from Salmonella typhimurium.
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Amino Acid Sequence↗
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Activity and thermostability of the small self-splicing group I intron in the pre-tRNA(lle) of the purple bacterium Azoarcus.
The 205-nt group I intron located in the pre-tRNA(lle) from the bacterium Azoarcus sp.BH72 is the smallest self-splicing group I intron identified to date. Comparative sequence analysis has placed this intron and the Anabaena pre-tRNA(Leu) intron into the same subgroup, IC3; we now compare their activity and stability. Unlike the Anabaena intron, the Azoarcus intron has two transitions in the kinetics of the first step of splicing. The faster transition occurs with a larger k(cat)/K(m) than that of the Anabaena or other group I introns, due to a rapid K(cat) (5 min(-1) at 32 degrees C) and a low K(m) for guanosine (17 microM). The excised intron circularizes by releasing a trinucleotide from the 5' end of the intron, another property unlike the Anabaena intron. Although it is smaller in size, the Azoarcus intron retains activity at higher temperatures, higher concentrations of urea, and higher pH than the Anabaena intron. Melting curves show that tertiary structure is disrupted at a lower temperature in the Anabaena intron. Some structural features that may explain the unusual stability of the Azoarcus intron include a G-C rich secondary structure and the presence of two 11-nt motifs, which are known to interact strongly with GAAA loops in group I and group II introns. The disruption of one of these interactions by substituting the Anabaena structural element in fact lowered the thermal stability of the Azoarcus intron. Thus, even superficially similar group I introns from the same structural subgroup can differ significantly in activity and stability.
Base Sequence↗
Modified constructs of the tRNA TPsiC domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases.
The TPsiC stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m(5)C(49), T(54), Psi(55)and m(1)A(58). U(54)is methylated to m(5)U (T) by m(5)U(54)methyltransferase (RUMT); A(58)is methylated to m(1)A by m(1)A(58)tRNA methyltransferase (RAMT). RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3'-half of the tRNA molecule. We report that RAMT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T(54)and Psi(55)), naturally occurring modifications at unnatural positions (m(5)C(60)), altered sugar puckers (dU(54)and/or dU(55)) or with disrupted U-turn interactions (m(1)Psi(55)or m(1)m(3)Psi(55)). The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T(54)increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate. Local conformation around U(54)was found to be an important determinant for the activities of both RAMT and RUMT.
Animals↗
RNA binding determinant in some class I tRNA synthetases identified by alignment-guided mutagenesis.
The N-terminal nucleotide binding folds of all 10 class I tRNA synthetases (RSs) contain characteristic conserved sequence motifs that define this class of synthetases. Sequences of C-terminal domains, which in some cases are known to interact with anticodons, are divergent. In the 676-amino acid Escherichia coli methionyl-tRNA synthetase (MetRS), interactions with the methionine tRNA anticodon are sensitive to substitutions at a specific location on the surface of the C-terminal domain of this protein of known three-dimensional structure. Although four class I synthetases of heterogeneous lengths and unknown structures are believed to be historically related to MetRS, pair-wise sequence similarities in the region of this RNA binding determinant are obscure. A multiple alignment of all sequences of three of these synthetases with all MetRS sequences suggested a location for the functional analog of the anticodon-binding site in these enzymes. We chose a member of this set for alignment-guided mutagenesis, combined with a functional analysis of mutant proteins. Substitutions within two amino acids of the site fixed by the multiple sequence alignment severely affected interactions with tRNA but not with ATP or amino acid. Multiple individual replacements at this location do not disrupt enzyme stability, indicating this segment is on the surface, as in the MetRS structure. The results suggest the location of an RNA binding determinant in each of these three synthetases of unknown structure.
Diphosphates↗
Efficient initiation of HIV-1 reverse transcription in vitro. Requirement for RNA sequences downstream of the primer binding site abrogated by nucleocapsid protein-dependent primer-template interactions.
Synthesis of HIV-1 (-) strong-stop DNA is initiated following annealing of the 3' 18 nucleotides (nt) of tRNA(3)(Lys) to the primer binding site (PBS) near the 5' terminus of viral RNA. Here, we have investigated whether sequences downstream of the PBS play a role in promoting efficient (-) strong-stop DNA synthesis. Our findings demonstrate a template requirement for at least 24 bases downstream of the PBS when tRNA(3)(Lys) or an 18-nt RNA complementary to the PBS (R18), but not an 18-nt DNA primer, are used. Additional assays using 18-nt DNA-RNA chimeric primers, as well as melting studies and circular dichroism spectra of 18-nt primer:PBS duplexes, suggest that priming efficiency is correlated with duplex conformation and stability. Interestingly, in the presence of nucleocapsid protein (NC), the 24 downstream bases are dispensable for synthesis primed by tRNA(3)(Lys) but not by R18. We present data supporting the conclusion that NC promotes extended interactions between the anticodon stem and variable loop of tRNA(3)(Lys) and a sequence upstream of the A-rich loop in the template. Taken together, this study leads to new insights into the initiation of HIV-1 reverse transcription and the functional role of NC-facilitated tRNA-template interactions in this process.
Base Sequence↗
Engineering mammalian aspartyl-tRNA synthetase to probe structural features mediating its association with the multisynthetase complex.
Aspartyl-tRNA synthetase from higher eukaryotes is a component of a multienzyme complex comprising nine aminoacyl-tRNA synthetases. The cDNA encoding cytoplasmic rat liver aspartyl-tRNA synthetase was previously cloned and sequenced. This work reports the identification of structural features responsible for its association within the multisynthetase complex. Mutant and chimeric proteins have been expressed in mammalian cells and their structural behavior analyzed. A wild-type rat liver aspartyl-tRNA synthetase, expressed in Chinese hamster ovary (CHO) cells, associates within the complex from CHO cells, whereas a mutant enzyme with a deletion of 34 amino acids from its amino-terminal extremity does not. A chimeric enzyme, made of the amino-terminal moiety of rat liver aspartyl-tRNA synthetase fused to the catalytic domain of yeast lysyl-tRNA synthetase, has been expressed in Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase. The fusion protein is stable in vivo, does not associate within the multisynthetase complex and cannot restore normal growth of the mutant cells. These results establish that the 3.7-kDa amino-terminal moiety of mammalian aspartyl-tRNA synthetase mediates its association with the other components of the complex. In addition, the finding that yeast lysyl-tRNA synthetase cannot replace the aspartyl-tRNA synthetase component of the mammalian complex, indicates that interactions between neighbouring enzymes also play a prominent role in stabilization of this multienzyme structure and strengthened the view that the multisynthetase complex is a discrete entity with a well-defined structural organization.
Amino Acid Sequence↗
Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA.
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Animals↗
Three-dimensional structure of tRNA.
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Base Sequence↗
Reconstitution of the biosynthetic pathway of selenocysteine tRNAs in Xenopus oocytes.
Selenocysteine is cotranslationally introduced into a growing polypeptide in response to certain UGA codons in selenoprotein mRNAs. The biosynthesis of this amino acid initiates by aminoacylation of specific tRNAs (designated tRNA([Ser]Sec)) with serine and subsequent conversion of the serine moiety to selenocysteine. The resulting selenocysteyl-tRNA then donates selenocysteine to protein. In most higher vertebrate cells and tissues examined, multiple selenocysteine isoacceptors have been described. Two of these have been determined to differ by only a single modified residue in the wobble position of the anticodon. In addition, the steady-state levels and relative distributions of these isoacceptors have been shown to be influenced by the presence of selenium. In order to gain a better understanding of the relationship between these tRNAs and how they are regulated, both the Xenopus selenocysteine tRNA gene and an in vitro synthesized RNA have each been injected into Xenopus oocytes and their maturation analyzed. In this system, selenium enhanced RNA stability and altered the distribution of isoacceptors that differ by a single ribose methylation. Interestingly, the biosynthesis of one of these modified nucleosides (5-methylcarboxymethyl-2'-O-methyluridine), which has been identified only in the wobble position of selenocysteine tRNA, also occurs in oocytes. Examination of the modified residues in both the naturally occurring Xenopus selenocysteine tRNA and the products generated from exogenous templates in oocytes demonstrated the faithful reconstruction of the biosynthetic pathway for these tRNAs.
Animals↗
tRNA's wobble decoding of the genome: 40 years of modification.
The genetic code is degenerate, in that 20 amino acids are encoded by 61 triplet codes. In 1966, Francis Crick hypothesized that the cell's limited number of tRNAs decoded the genome by recognizing more than one codon. The ambiguity of that recognition resided in the third base-pair, giving rise to the Wobble Hypothesis. Post-transcriptional modifications at tRNA's wobble position 34, especially modifications of uridine 34, enable wobble to occur. The Modified Wobble Hypothesis proposed in 1991 that specific modifications of a tRNA wobble nucleoside shape the anticodon architecture in such a manner that interactions were restricted to the complementary base plus a single wobble pairing for amino acids with twofold degenerate codons. However, chemically different modifications at position 34 would expand the ability of a tRNA to read three or even four of the fourfold degenerate codons. One foundation of Crick's Wobble Hypothesis was that a near-constant geometry of canonical base-pairing be maintained in forming all three base-pairs between the tRNA anticodon and mRNA codon on the ribosome. In accepting an aminoacyl-tRNA, the ribosome requires maintenance of a specific geometry for the anticodon-codon base-pairing. However, it is the post-transcriptional modifications at tRNA wobble position 34 and purine 37, 3'-adjacent to the anticodon, that pre-structure the anticodon domain to ensure the correct codon binding. The modifications create both the architecture and the stability needed for decoding through restraints on anticodon stereochemistry and conformational space, and through selective hydrogen bonding. A physicochemical understanding of modified nucleoside contributions to the tRNA anticodon domain architecture and its decoding of the genome has advanced RNA world evolutionary theory, the principles of RNA chemistry, and the application of this knowledge to the introduction of new amino acids to proteins.
Anticodon↗
Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe).
The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement.
Base Sequence↗
Synthesis, modifications, and turnover of proteins during aging.
Slowing down of bulk protein synthesis is one of the most commonly observed biochemical changes during aging. The implications and consequences of slower rates of protein synthesis are manifold, including a decrease in the availability of enzymes for the maintenance, repair, and normal metabolic functioning of the cell, an inefficient removal of inactive, abnormal, and damaged macromolecules in the cell, the inefficiency of the intracellular and intercellular signalling pathways, and a decrease in the production and secretion of hormones, antibodies, neurotransmitters, and the components of the extracellular matrix. Age-related changes in the activity, specificity, and stability of a large number of proteins have been reported. However, the molecular mechanisms responsible for such alterations are still poorly understood. Studies on various components of the protein synthetic machinery have revealed a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors. Because posttranslational modifications of proteins determine their activity and stability, alterations in the extent and level of various modifications such as phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and conformational changes during aging are being studied. Changes in the regulation of protein synthesis, posttranslational modifications, and protein turnover are crucial determinants of age-related decline in the maintenance, repair, and survival of the organism.
Adenosine Diphosphate Ribose↗
Regulation of protein synthesis in rabbit reticulocyte lysates: additional initiation factor required for formation of ternary complex (eIF-2.GTP.Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase.
Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.
Animals↗
Functional defects of pathogenic human mitochondrial tRNAs related to structural fragility.
Aminoacylation of transfer RNAs (tRNAs) is essential for protein synthesis. A growing number of human diseases correlate with point mutations in tRNA genes within the mitochondrial genome. These tRNAs have unique sequences that suggest they have fragile structures. However, the structural significance of pathology-related tRNA mutations and their effects on molecular function have not been explored. Here, opthalmoplegia related mutants of a human mitochondrial tRNA have been investigated. Each mutation replaces either an A-U or G-C pair in the predicted secondary structure with an A-C pair. Aminoacylation of each mutant tRNA was severely attenuated. Moreover, each strongly inhibited aminoacylation of the wild type substrate, suggesting that the effects of these mutations might not be bypassed in the potentially heteroplasmic environment of mitochondria. The function of mutant tRNAs was rescued by single compensatory mutations that restored Watson-Crick base pairing and reintroduced stability into regions of predicted secondary structure, even though the pairs introduced were different from those found in the wild type tRNA. Thus, functional defects caused by a subset of pathogenic mutations may result from the inherent structural fragility of human mitochondrial tRNAs.
Humans↗
Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.
Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.
Anti-Bacterial Agents↗
Efficient introduction of aryl bromide functionality into proteins in vivo.
Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20-25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.
Amino Acid Substitution↗
Mutagenesis of Arg335 in bovine mitochondrial elongation factor Tu and the corresponding residue in the Escherichia coli factor affects interactions with mitochondrial aminoacyl-tRNAs.
During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of the ribosome. Mammalian mitochondrial EF-Tu (EF-Tu(mt)) carries out this activity using aa-tRNAs that lack many of the invariant or semi-invariant residues that stabilize the 3-dimensional structures of canonical tRNAs. The primary sequence of EF-Tu is highly conserved. However, several residues involved in aa-tRNA binding are not conserved between the mitochondrial and bacterial factors. One such residue, located at position 287 in Escherichia coli EF-Tu, is adjacent to the 5' end of the aa-tRNA and is acidic in all prokaryotic factors but is basic in EF-Tu(mt). Site-directed mutagenesis of this residue (Glu287) in E. coli EF-Tu and complementary mutagenesis of the corresponding Arg335 in EF-Tu(mt) was performed to create E. coli EF-Tu E287R and EF-Tu(mt) R335E respectively. EF-Tu(mt) R335E has a reduced activity in ternary complex formation and A-site binding with mitochondrial Phe-tRNA.(Phe) In contrast, E. coli EF-Tu E287R is more active that the wild-type factor in forming ternary complexes with mitochondrial Phe-tRNA,(Phe) and the variant promotes the binding of mitochondrial aa-tRNA to the ribosome more effectively than does the wild-type factor. Both EF-Tu(mt) R335E and E. coli EF-Tu E287R have activities comparable to the corresponding wild-type factors in assays using E. coli Phe-tRNA.(Phe) These data suggest that the residue at position 287 plays an important role in the binding and EF-Tu-mediated delivery of mitochondrial aa-tRNAs to the A-site of the ribosome.
Amino Acid Sequence↗
Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria.
Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The "21st synthetase-tRNA pairs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.
Amino Acids↗