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PubMed2002

Group I introns and RNA folding.

Before the discovery of catalytic RNA, tRNA molecules were the most studied RNA molecules for understanding RNA folding. Afterwards, group I introns, because of their stability and the fact that structural folding could be monitored by following their catalytic activity, became the molecule of choice for studying RNA architecture and folding. A major advantage of group I introns for studying the catalytic activity of RNA molecules is that catalytic activity is triggered by the addition of external guanosine cofactors. The self-splicing activity can therefore be precisely controlled. Using group I introns, several RNA motifs central to RNA-RNA self-assembly and folding were discovered. The analysis of the recent X-ray structures of the rRNA subunits indicates that several motifs present in the ribosome occur also in various group I introns.

Base Sequence↗
PubMed1976

The kinetics of binding of U-U-C-A to a dodecanucleotide anticodon fragment from yeast tRNA-Phe.

The kinetics of U-U-C-A binding to the dodecanucleotide (A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-Gp) isolated from the anticodon region of yeast tRNA-Phe are similar to the kinetics of binding of U-U-C-A to intact tRNA-Phe. A large enhancement in binding constant over that predicted for U-U-C-A-U-G-A-A is observed for both the complexes of dodecanucleotide and tRNA-Phe with U-U-C-A. This strongly suggests that both the anticodon loop in tRNA-Phe and the dodecanucleotide can form four base pairs with U-U-C-A. Furthermore, the enhanced stability cannot be attributed to a special conformation of the anticodon loop, but instead the anticodon loop is probably flexible. A likely explanation for the increased binding is the effect of non-base-paired ends. This increased thermodynamic stability comes from a larger entropy gain rather than a larger enthalpy decrease.

Adenine Nucleotides↗
PubMed1990

The conformation of single-stranded nucleic acids tDNA versus tRNA.

Conformational analyses using the single-strand-specific nuclease from mung bean and restriction endonucleases have been performed on a series of DNA fragments related to the sequence of the yeast initiator tRNA(Met). Mung bean nuclease cleaves DNA fragments exclusively in some, but not all, single-stranded regions as predicted by RNA secondary structural rules. Comparison of cleavage patterns of yeast initiator tRNA(Met), tDNA(Met) (a DNA oligomer having the sequence of tRNA(Met] and the anti-tDNA(Met) (the complement of tDNA(Met] suggests that the conformation of the three molecules is very similar. Furthermore, both tDNA and anti-tDNA are cleaved by HhaI and CfoI restriction endonucleases at two GCG/C sites which would be in double-stranded regions (the acceptor and dihydrouridine stem), if the two molecules adopt the tRNA cloverleaf structure. On the other hand, minor cleavage products show that the core region, i.e. the extra loop area, is slightly more exposed in tDNA and in anti-tDNA than in tRNA. Therefore, we submit that the global conformation of nucleic acids is primarily dictated by the interaction of purine and pyrimidine bases with atoms and functional groups common to both RNA and DNA. In this view the 2'-hydroxyl group, in tRNA at least, is an auxiliary structural feature whose role is limited to fostering local interactions, which increase the stability of a given conformation.

Base Sequence↗
PubMed1978

The effects of trimethoprim and erythromycin on polysome metabolism in Escherichia coli.

Trimethoprim and erythromycin were shown to have different overall effects on in vivo polysome metabolism in Escherichia coli. In a rel A+-rel A pair of strains, trimethoprim treatment induces a reduction of polysome level to a variable extent, similarly to aminoacyl tRNA deprivation of cells, but persisting polysomes remain dynamic structures in a state of continual turnover. In contrast, erythromycin stabilizes polysome level to a high value in either kind of strain, but maintained polysomes appear as "frozen" structures unable to undergo ribosome translocation.

Erythromycin↗
PubMed1993

The discriminator base influences tRNA structure at the end of the acceptor stem and possibly its interaction with proteins.

For many tRNAs, the discriminator base preceding the CCA sequence at the 3' end is important for aminoacylation. We show that the discriminator base influences the stability of the 1.72 base pair onto which it is stacked. Mutations of the discriminator base from adenosine to cytidine or uridine make the cytidine residue in the C1-G72 base pair of mutant Escherichia coli initiator tRNAs more reactive toward sodium bisulfite, the single-strand-specific reagent. The activity of the enzyme Met-tRNA transformylase toward these and other mutant initiator tRNAs is also consistent with destabilization of the 1.72 base pair in vitro and in vivo. By influencing the strength of the 1.72 base pair, the discriminator base could affect the energetic cost of opening the base pair and modulate the structure of the tRNA near the site of aminoacylation. For some aminoacyl-tRNA synthetases and other proteins that interact with tRNA, these factors could be important for specific recognition and/or formation of the transition state during catalysis.

Acyltransferases↗
PubMed1976

Interaction of elongation factor 1 with aminoacylated brome mosaic virus and tRNA's.

Tyrosylated Brome mosaic virus RNA was found to interact with a binary complex of wheat germ, elongation factor 1 and [3H]GTP. Increasing amounts of the aminoacylated viral RNA proportionately reduced radioactivity bound to a nitrocellulose filter, as has previously been noted by others for the charged forms of tobacco mosaic virus, turnip yellow mosaic virus, and tRNA's. However, Sephadex chromatography of the products showed that instead of forming the ternary complex elongation factor-GTP-aminoacyl RNA, the viral RNA caused release of GTP from its complex with elongation factor. Acetylated tyrosyl Brome mosaic virus RNA did not react with the binary complex,and only a slight degree, if any, of stabilization of tyrosine bound to viral RNA was observed after interaction with elongation factor 1. Although such interactions are similar to the reaction of elongation factor with aminoacyl-tRNA , the release of GTP is different and accentuates the possible role for aminoacylation in transcription rather than in translation events.

Guanosine Triphosphate↗
PubMed1987

Construction and analysis of deletions in the amino-terminal extension of glutamine tRNA synthetase of Saccharomyces cerevisiae.

GLN4 of Saccharomyces cerevisiae encodes an amino-terminal extension of 224 amino acids. This is connected to a polypeptide which is colinear with and 40% identical to Escherichia coli glutamine tRNA synthetase. We examined the potential significance of the amino-terminal extension. Two single base and five multiple base frame shift deletions were constructed in this segment. Each of these mutations is associated with a lethal phenotype. This suggests that the coding sequence for the amino-terminal extension is translated. It also implies that there are no translation restarts downstream of the coding region for the amino-terminal extension which produce active enzyme. Three internal deletions of various sizes, and which preserve the correct reading frame, were constructed in the coding region of the amino-terminal extension. Cells which harbor such in-frame deletions on a multi copy plasmid are viable, even when a deletion construct is the only source of GLN4-encoded activity. Extracts of cells which have one of these deletions have reduced, but measurable, glutamine tRNA synthetase activity. We conclude that the catalytic activity resides with the segment which is homologous to the E. coli enzyme and that the amino-terminal extension itself is dispensable for aminoacylation activity. Each of the internal in-frame deletion constructions is respiration-proficient. The amino-terminal extension, therefore, is not used for an essential mitochondrial function of the GLN4 gene product. Within the accuracy of the measurements, activities of four other aminoacyl-tRNA synthetases are not affected by the presence of a GLN4 internal deletion allele as the only source of GLN4-encoded activity. This suggests that the amino-terminal extension does not stabilize a complex which includes one or more of these four enzymes and whose activity depends on proper assembly of the complex.

Alleles↗
PubMed1998

Molecular instability in the COII-tRNA(Lys) intergenic region of the human mitochondrial genome: multiple origins of the 9-bp deletion and heteroplasmy for expanded repeats.

We have identified two individuals from Glasgow in Scotland who have a deletion of one of two copies of the intergenic 9-bp sequence motif CCCCCTCTA, located between the cytochrome oxidase II (COII) and lysine tRNA (tRNA(Lys)) genes of the human mitochondrial genome. Although this polymorphism is common in Africa and Asia, it has not been reported in Northern Europe. Analysis of the mitochondrial DNA control region sequences of these two individuals suggests that they belong to a lineage that originated independently of the previously characterized African and Asian 9-bp deleted lineages. Among the Scottish population we have also identified a maternal lineage of three generations exhibiting heteroplasmy for two, three and four copies of the CCCCCTCTA motif. Polymerase chain reaction amplification across the COII-tRNA(Lys) intergenic region of these individuals gives different ratios of the three product lengths that are dependent on the concentration of the DNA-binding dye crystal violet. To investigate whether changes in repeat number were generated de novo, we constructed clones containing known numbers of the CCCCCTCTA motif. In the presence of high concentrations of crystal violet we obtained two, three and four copies of this motif when the amplification template contained only four copies. Various DNA-binding drugs are known to stabilize bulged structures in DNA and contribute to the process of slipped-strand mispairing during DNA replication. These results suggest that the COII-tRNA(Lys) intergenic region is unstable owing to slipped-strand mispairing. Although sequences containing four copies of the CCCCCTCTA motif are less stable in vitro, we observed an increase in the proportion of mitochondrial genomes with four repeats between-a mother and a daughter in the heteroplasmic lineage. From this we conclude that drift in the germ-line lineage is a main factor in the maintenance or loss of heteroplasmy.

Africa↗
PubMed1997

The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase.

The crystal structure of an enzyme-substrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme-product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl2 reveal the existence of two metal binding sites between beta- and gamma-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.

Acylation↗
PubMed2000

Construction of a ribozyme-expression system that effectively transports ribozymes to the cytoplasm.

In our previous studies, it was demonstrated that the activity of a ribozyme in vivo was governed by several parameters, which include a high level-expression of ribozyme, the intracellular stability of the ribozyme and colocalization of the ribozyme with its target RNA in the same cellular compartment. To generate ribozymes with significant activity in vivo, we have developed a ribozyme-expression system based on a human tRNA(Val) promoter. Our tRNA-embedded ribozymes produced by our ribozyme-expression system remain relatively stable in cultured cells with half-lives longer than 30 min. Moreover, tRNA-ribozymes with a cloverleaf structure were efficiently exported from the nucleus to the cytoplasm, where they would effectively cleave target RNAs. In the present study, we investigated the relationship between the secondary structure of the tRNA-ribozymes and the transport efficacy of them in mammalian cells by using a screening system in vivo. Furthermore, we also investigated the mechanism of the export of tRNA-embedded ribozymes both in mammalian cells and in Xenopus oocytes.

Animals↗
PubMed1999

Single atom modification (O-->S) of tRNA confers ribosome binding.

Escherichia coli tRNALysSUU, as well as human tRNALys3SUU, has 2-thiouridine derivatives at wobble position 34 (s2U*34). Unlike the native tRNALysSUU, the full-length, unmodified transcript of human tRNALys3UUU and the unmodified tRNALys3UUU anticodon stem/loop (ASLLys3UUU) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNAPhe anticodon stem/loop (ASLPheGAA) had an affinity (Kd = 136+/-49 nM) similar to that of native yeast tRNAPheGmAA (Kd = 103+/-19 nM). We have found that the single, site-specific substitution of s2U34 for U34 to produce the modified ASLLysSUU was sufficient to restore ribosomal binding. The modified ASLLysSUU bound the ribosome with an affinity (Kd = 176+/-62 nM) comparable to that of native tRNALysSUU (Kd = 70+/-7 nM). Furthermore, in binding to the ribosome, the modified ASLLys3SUU produced the same 16S P-site tRNA footprint as did native E. coli tRNALysSUU, yeast tRNAPheGmAA, and the unmodified ASLPheGAA. The unmodified ASLLys3UUU had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASLLys3SUU was different from that of the unmodified ASLLysUUU, whereas the stems were isomorphous. Based on these and other data, we conclude that s2U34 in tRNALysSUU and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U34-->s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA.

Aldehydes↗
PubMed1999

In vivo and in vitro processing of the Bacillus subtilis transcript coding for glutamyl-tRNA synthetase, serine acetyltransferase, and cysteinyl-tRNA synthetase.

In Bacillus subtilis, the adjacent genes gltX, cysE, and cysS encoding respectively glutamyl-tRNA synthetase, serine acetyl-transferase, and cysteinyl-tRNA synthetase, are transcribed as an operon but a gltX probe reveals only the presence of a monocistronic gltX mRNA (Gagnon et al., 1994, J Biol Chem 269:7473-7482). The transcript of the gltX-cysE intergenic region contains putative alternative secondary structures forming a p-independent terminator or an antiterminator, and a conserved sequence (T-box) found in the leader of most aminoacyl-tRNA synthetase and many amino acid biosynthesis genes in B. subtilis and in other Gram-positive eubacteria. The transcription of these genes is initiated 45 nt upstream from the first codon of gltX and is under the control of a sigmaA-type promoter. Analysis of the in vivo transcript of this operon revealed a cleavage site immediately downstream from the p-independent terminator structure. In vitro transcription analysis, using RNA polymerases from Escherichia coli, B. subtilis, and that encoded by the T7 phage, in the presence of various RNase inhibitors, shows the same cleavage. This processing generates mRNAs whose 5'-end half-lives differ by a factor of 2 in rich medium, and leaves putative secondary structures at the 3' end of the gltX transcript and at the 5' end of the cysE/S mRNA, which may be involved in the stabilization of these mRNAs. By its mechanism and its position, this cleavage differs from that of the other known transcripts encoding aminoacyl-tRNA synthetases in B. subtilis.

Acetyltransferases↗
PubMed1994

The invariant arginine in motif 2 of Escherichia coli alanyl-tRNA synthetase is important for catalysis but not for substrate binding.

Structural motifs 2 and 3, located in the active site of class II aminoacyl-tRNA synthetases, each contain an invariant arginine thought to participate in interactions with ATP. For Escherichia coli alanyl-tRNA synthetase (AlaRS), sequence comparisons indicate that Arg-69 should be aligned with the invariant arginine in motif 2 of other class II synthetases. Site-directed random mutagenesis has been employed to generate a set of proteins containing amino acid substitutions in a portion of motif 2 of AlaRS. In this set, only mutations at position 69 caused the enzyme to lose ability to complement growth of an alaS deletion strain, and proteins containing substitutions at position 69 alone are undetectable in a Western blot assay. A mutant protein containing the transposition of Arg-69 with Gly-71 does not complement growth but does accumulate in vivo and has thus been purified. Michaelis and dissociation constants for the interaction of this protein with ATP are indistinguishable from those of the wild-type enzyme. However, this two-position displacement of the arginine causes a decrease in the kcat for the ATP-PPi exchange reaction by 2 orders of magnitude. These data suggest a role for the invariant arginine of motif 2 in stabilization of the transition state during alanyladenylate synthesis.

Alanine-tRNA Ligase↗
PubMed1987

A thermostable Gm-methylase recognizes the tertiary structure of tRNA.

The efficiency of methylation of tRNA by a thermostable tRNA(guanosine-2')-methyltransferase (Gm-methylase) was examined at various temperatures using several species of tRNA isolated from Escherichia coli, yeast and Bacillus subtilis, each possessing different thermal properties. The optimal temperature for the methylation reaction was ca. 20 degrees C lower than the melting temperature of the tRNA in each case. Arrhenius plots of the methylation reactions with various tRNAs gave straight lines below the optimal temperatures in all cases, with similar activation energies of between 10 and 14 kcal/mol. Above the optimal temperatures, the methyl acceptor activity decreased as the incubation temperature was raised to 80 degrees C, at which point the methylase was still active. A correlation was observed between the remaining methyl acceptor activity and the hyperchromicity of tRNA. These results suggest that Gm-methylase recognizes the tertiary structure of tRNA, and it is not the substrate tRNA but the enzyme which is activated by heat.

Base Sequence↗
PubMed1998

Derivation of the three-dimensional architecture of bacterial ribonuclease P RNAs from comparative sequence analysis.

The secondary structure of bacterial RNase P RNA, a ribozyme responsible for the maturation of the 5' end of tRNAs, is well established on the basis of sequence comparison analysis. RNase P RNA secondary structures fall into two types, A and B, which share a common core formed by the assembly of two main folding domains, but differ in their peripheral elements.A revised alignment of 137 available sequences reveals new covariations allowing for the refinement of both types of secondary structures. Phylogenetic evidence is thus provided for the extension of stems P11, P14, P19, P10.1 and P15.1 through further canonical base-pairs or GAellipsisGA mismatches. These refinements led in turn to a new organization of the catalytic core, with coaxial stackings of helices P2 and P19 as well as P1 and P4. New inter-domain tertiary interactions involve loop L9 and helix P1 and loop L8 with helix P4. These features were incorporated into atomic-scale 3D models of RNase P RNA for representatives of each structural type, namely Escherichia coli and Bacillus subtilis. In each model, the juxtaposition of the core helices creates a cradle onto which the pre-tRNA substrate binds with most evolutionarily conserved residues converging towards the cleavage site. The inner cores of both types are stabilized similarly, albeit by different peripheral elements, emphasizing the modular and hierarchical organisation of the architecture of RNase P RNAs. Similarities are thus apparent between the type A modules, P16/P17/P6 and P13/P14, and their type B analogs, P5.1/P15.1 and P10. 1/P10.1a, respectively. Other noteworthy features of these models include compactness and good agreement with published crosslinking data.

Bacteria↗
PubMed2002

The quaternary structure of the HisZ-HisG N-1-(5'-phosphoribosyl)-ATP transferase from Lactococcus lactis.

The N-1-(5'-phosphoribosyl)-ATP transferase (ATP-PRTase) encoded by the hisG locus catalyzes the condensation of ATP with PRPP, the first reaction in the biosynthesis of histidine. Unlike the homohexameric forms of the enzyme found in Escherichia coli and Salmonella typhimurium, the ATP-PRTase from Lactococcus lactis and a number of other bacterial species consists of two different polypeptides, both of which are required for catalytic activity (Sissler et al. (1999) Proc. Natl. Acad. Sci. 96, 8985-8990). The first of these is a truncated version of HisG that is approximately 100 amino acids shorter than the canonical versions. The second, HisZ, is a 328-residue version of a class II aminoacyl-tRNA synthetase catalytic domain that possesses no aminoacylation function. Here, the molecular mass and subunit composition of the L. lactis HisZ-HisG heteromeric ATP-PRTase is investigated using liquid chromatography, analytical ultracentrifugation, and quantitative protein sequencing. Individually, HisZ and HisG form inactive but stable dimers with association constants in the range of 2.5-3.3 x 10(5) M(-1). When both types of subunits are present, a quaternary octamer complex is formed with a sedimentation coefficient of 10.1 S. Incubation of this complex with ATP promotes a shift to 10.7 S. By contrast, incubation with the allosteric modulators AMP and histidine destabilizes the complex, resulting in a shift to multiple species in equilibrium with an average of 9.3 S. While this octameric structure is unique to both the phosphoribosyl transferases and the aminoacyl-tRNA synthetases, the change in sedimentation behavior elicited by substrates and inhibitors suggests the presence of allosteric regulatory mechanisms reminiscent of other multisubunit enzymes of metabolic importance.

Amino Acyl-tRNA Synthetases↗