Modified nucleoside, 5-carbamoylmethyluridine, located in the first position of the anticodon of yeast valine tRNA

A novel modified nucleoside located in the first position of the anticodon of yeast tRNAVal2a was isolated and its chemical structure was characterized as 5-carbamoylmethyluridine by means of ultraviolet absorption spectrum, mass spectrum, and nuclear magnetic resonance spectrum.     

Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

A modified nucleoside located in the anticodon of proline tRNA from Torulopsis utilis is 5-carbamoylmethyluridine

A modified nucleoside has been isolated from the first position of the anticodon of Torulopsis utilis tRNAPro. It was identified to be an uridine derivative, 5-carbamoylmethyluridine from analyses of its UV, 1H-NMR, and secondary ion mass spectra.     

Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36). By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme. A 17-base oligoribonucleotide corresponding to the anticodon stem-loop of E. coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave k(cat)s similar to that for the normal substrate (full-length tRNA(Phe) unmodified at A37), although the K(m) for minihelix(Phe) was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification. Changing A36 to G in the minihelix resulted in a 260-fold reduction in k(cat) compared to minihelix(Phe) and a 13-fold increase in K(m). An A38G variant was modified with a 9-fold reduction in k(cat) and a 5-fold increase in K(m). A random coil 17-base oligoribonucleotide in which the loop sequence of E. coli tRNA(Phe) was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix-loop structure is essential for recognition. Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region.     

Identification of a modified nucleoside located in the first position of the anticodon of Torulopsis utilis tRNAPro as 5-carbamoylmethyluridine

An unknown nucleoside in the first position of the anticodon of Torulopsis utilis tRNAPro has been isolated. The UV, 1H NMR and secondary ion mass spectra indicated that this nucleoside is a uridine derivative, 5-carbamoylmethyluridine. The structure was completely established by comparison of the instrumental analysis results and chromatographic behavior of the isolated nucleoside with those of a synthetic sample.     

Identification and codon reading properties of 5-cyanomethyl uridine,a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA₂^Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA₂^Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.     

Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli.

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.     

Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl-tRNA synthetase.

Cytidine in the anticodon second position (position 35) and G or U in position 36 of tRNAArg are required for aminoacylation by arginyl-tRNA synthetase (ArgRS) from Escherichia coli. Nevertheless, an arginine-accepting amber suppressor tRNA with a CUA anticodon (FTOR1Delta26) exhibits suppression activity in vivo [McClain, W.H. & Foss, K. (1988) Science, 241, 1804-1807]. By an in vitro kinetic study with mutagenized tRNAs, we showed that the arginylation of FTOR1Delta26 involves C34 and U35, and that U35 can be replaced by G without affecting the activity. Thus, the positioning of the essential nucleotides for the arginylation is shifted to the 5' side, by one residue, in the suppressor tRNAArg. We found that the shifted positioning does not depend on the tRNA sequence outside the anticodon. Furthermore, by a genetic method, we isolated a mutant ArgRS that aminoacylates FTOR1Delta26 more efficiently than the wild-type ArgRS. The isolated mutant has mutations at two nonsurface amino-acid residues that interact with each other near the anticodon-binding site.     

Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli tRNA

A selenium-containing nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is present in lysine- and glutamate-isoaccepting tRNA species of Escherichia coli. The synthesis of mnm5se2U is optimum (4 mol/100 mol tRNA) when selenium is present at about 1 microM concentration and is neither decreased by a high (8 mM) level of sulfur in the medium nor increased by excessive (10 or 100 microM) levels of selenium. Lysine- and glutamate-isoaccepting tRNA species that contain 5-methylaminomethyl-2-thiouridine (mnm5s2U) coexist with the seleno-tRNAs in E. coli cells and a reciprocal relationship between the mnm5se2U- and the mnm5s2U-containing species is maintained under a variety of growth conditions. The complete 5-methylaminomethyl side chain is not a prerequisite for introduction of selenium at the 2-position. In E. coli mutants deficient in the ability to synthesize the 5-methylaminomethyl substituent, both the 2-thiouridine and the corresponding 2-selenouridine derivatives of intermediate forms are accumulated. Broken cell preparations of E. coli synthesize mnm5se2U in tRNAs by an ATP-dependent process that appears to involve the replacement of sulfur in mnm5s2U with selenium.