The chemical synthesis of E. coli tRNA(Lys) anticodon loop fragment and its analogues

E. coli tRNA(Lys) anticodon loop fragment (Umnm5s2UUUt6A) 1 and its analogues 2-6 were synthesized by the classical phosphotriester approach in solution. The preparation of suitably protected derivatives of N6-threonylcarbamoyladenosine 18 is also described.     

Cross-species aminoacylation of tRNA with a long variable arm between Escherichia coli and Saccharomyces cerevisiae

Prokaryotes have three amino acid-specific class II tRNAs that possess a characteristic long variable arm, tRNASer, tRNALeuand tRNATyr, while eukaryotes have only two, tRNASerand tRNALeu. Because of such a phylogenetic divergence in the composition of tRNA, the class II tRNA system is a good candidate for studying how the tRNA recognition manner has evolved in association with the evolution of tRNA. We report here a cross-species aminoacylation study of the class II tRNAs, showing the unilateral aminoacylation specificity between Escherichia coli and a yeast, Saccharomyces cerevisiae. Both SerRS and LeuRS from E.coli were unable to aminoacylate yeast class II tRNAs; in contrast, the yeast counterparts were able to aminoacylate E.coli class II tRNAs. Yeast seryl-tRNA synthetase was able to aminoacylate not only E.coli tRNASerbut also tRNALeuand tRNATyr, and yeast LeuRS was able to aminoacylate not only E.coli tRNALeubut also tRNATyr. These results indicate that the recognition manner of class II tRNA, especially the discrimination strategy of each aminoacyl-tRNA synthetase against noncognate class II tRNAs, is significantly divergent between E.coli and yeast. This difference is thought to be due mainly to the different composition of class II tRNAs in E.coli and yeast.     

Microhelix aminoacylation by a class I tRNA synthetase. Non-conserved base pairs required for specificity

Nucleotides in tRNAs that are conserved among isoacceptors are typically considered as candidates for tRNA synthetase recognition, with less importance attached to non-conserved nucleotides. Although the anticodon is an important contributor to the identity of methionine tRNAs, the class I methionine tRNA synthetase aminoacylates microhelices with high specificity. The microhelix substrates are comprised of as few as the 1st 4 base pairs of the acceptor stems of the elongator and initiator methionine tRNAs. For these two tRNAs, only the central 2:71 and 3:70 base pairs are common to the 1st 4 acceptor stem base pairs. We show here that, although the flanking 4:69 base pair is not conserved, a particular substitution at this position substantially reduces the gel electrophoresis-detected aminoacylation of an acceptor stem substrate that has the conserved 2:71 and 3:70 base pairs. Although the two methionine tRNAs have either U:A or G:C at position 4:69, substitution with C:G reduces charging of 9- or 4-base pair substrates that recreate part or all of the acceptor stem of a methionine tRNA. This effect is sufficient for methionine tRNA synthetase to discriminate between the closely related methionine and isoleucine tRNA acceptor stems. The ability to distinguish G:C and U:A from C:G is contrary to a simple scheme for recognition of atoms in the RNA minor groove.     

Modified nucleosides in the first positions of the anticodons of tRNA(Leu)4 and tRNA(Leu)5 from Escherichia coli

Minor leucine tRNA species, tRNA(Leu)4 and tRNA(Leu)5, from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mol. Biol. 129, 567-585]. In the present study, these two tRNA(Leu) species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA(Leu)5 was found to correspond to the tRNA gene reported as su degrees6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mol. Biol. 177, 627-644]. The first letter of the anticodon was identified to be 2'-O-methylcytidine (Cm). tRNA(Leu)4 was identified as the minor leucine tRNA that has been sequenced previously (tRNA(Leu)UUR) [Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220-2225]. There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA(Leu)4. Nucleoside N* was isolated to homogeneity (1 A260 unit). By 1H NMR spectroscopy, nucleoside N was found to be a 2'-O-methyluridine derivative with a substituent having a -CH2NH2+CH2COO- moiety in position 5 of the uracil ring. On the basis of these NMR analyses together with mass spectrometry, the chemical structure of nucleoside N* was determined as 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um). Nucleoside N* was thus found to be a novel type of naturally occurring modified uridine. Because of the conformational rigidity of Cm and cmnm5Um in the first position of the anticodon, these tRNA(Leu) species recognize the leucine codons UUA++ and UUG correctly, but never recognize the phenylalanine codons UUU and UUC.     

Maize mitochondrial seryl-tRNA synthetase recognizes Escherichia coli tRNA(Ser) in vivo and in vitro

After ovulation in salmonids, the eggs are held in the peritoneal cavity and bathed in coelomic fluid. Using a chromogenic peptide substrate, the anti-protease activity of brook trout coelomic fluid was measured. Trypsin, chymotrypsin, and pancreatic elastase activities were significantly inhibited by coelomic fluid containing 5.0, 10.0, and 25.0 microgram of total protein, respectively. Using subtractive cDNA cloning, we have previously characterized a set of ovarian proteins called TOPs (trout ovulatory proteins) that are secreted into the coelomic fluid after ovulation. TOPs are most homologous to mammalian antileukoprotease, a heat- and acid-stable serine protease inhibitor. On the basis of this homology, we hypothesized that the anti-trypsin activity observed in the coelomic fluid was related to the presence of TOPs. In the present study, this hypothesis was supported by the acid- and heat-stability of the anti-trypsin activity present in coelomic fluid. Coelomic fluid could be heated to 50 degrees C or treated at a pH less than 5.2 without a significant decrease in the inhibitory activity. Further, coelomic fluid from which TOPs were immunoprecipitated had significantly less anti-trypsin activity than nonimmunoprecipitated controls. We propose that TOP proteins are uniquely produced by the ovary and secreted into the coelomic fluid to act as protease inhibitors following ovulation.     

NMR studies of the effects of the 5'-phosphate group on conformational properties of 5-methylaminomethyluridine found in the first position of the anticodon of Escherichia coli tRNA(Arg)4

5-Methylaminomethyluridine (mnm5U) exists in the first position of the anticodon (position 34) of Escherichia coli tRNA4Arg for codons AGA/AGG. In the present study, the temperature dependence of the ribose-puckering equilibrium of pmnm5U was analyzed by proton NMR spectroscopy. Thus, the enthalpy difference (delta H) between the C2'-endo and C3'-endo forms was obtained at 0.65 kcal.mol-1. By comparison of the delta H values of pU and pmnm5U, the 5-substitution was found to increase the relative stability of the C3'-endo form over the C2'-endo form significantly (by 0.56 kcal.mol-1). Furthermore, this conformational "rigidity" was concluded to depend on the 5'-phosphate group, because nucleoside U exhibits only a negligible change in the ribose-puckering equilibrium upon the 5-methylaminomethyl substitution. Further NMR analyses and molecular dynamics calculations revealed that interactions between the 5-methylaminomethyl and 5'-phosphate groups of pmnm5U restrict the conformation about the glycosidic bond to a low anti form, enhancing steric repulsion between the 2-carbonyl and 2'-hydroxyl groups in the C2'-endo form. This intrinsic conformational rigidity of the mnm5U residue in position 34 may contribute to the correct codon recognition.     

Glutamate-459 is important for Escherichia coli branching enzyme activity

The branching enzyme belongs to the amylolytic family, a group of enzymes that cleave and/or transfer chains of glucan. The amylolytic enzymes are homologous and all contain four conserved regions, proposed to contain the active site. By primary structure analysis, a conserved position unique to branching enzymes has been identified. This residue, which is either Asp or Glu, depending on the species, is located immediately after the putative catalytic Glu-458 (Escherichia coli numbering). Branching enzymes differ from other amylolytic enzymes in having this acid pair, and we asked if this motif could be essential for branching enzyme action. We used site-directed mutagenesis of the Glu-459 residue in the E. coli branching enzyme in order to determine the significance of the conserved Asp/Glu in branching enzymes. A substitution of Glu-459 to Asp resulted in increased specific activity compared to wild-type, suggesting that the mutation had created a more efficient enzyme. Changing Glu-459 to Ala, Lys, or Gln lowered the specific activities and altered the preferred substrate from amylose to amylopectin.     

Crystal structures of three misacylating mutants of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP

Three previously described mutant Escherichia coli glutaminyl-tRNA synthetase (GlnRS) proteins that incorrectly aminoacylate the amber suppressor derived from tRNATyr (supF) with glutamine were cocrystallized with wild-type tRNAGln and their structures determined. In two of the mutant enzymes studied, Asp235, which contacts base pair G3-C70 in the acceptor stem, has been changed to asparagine in GlnRS7 and to glycine in GlnRS10. These mutations result in changed interactions between Asn235 of GlnRS7 and G3-C70 of the tRNA and an altered water structure between Gly235 of GlnRS10 and base pair G3-C70. These structures suggest how the mutant enzymes can show only small changes in their ability to aminoacylate wild-type cognate tRNA on the one hand and yet show a lack of discrimination against a noncognate U3-A70 base pair on the other. In contrast, the change of Ile129 to Thr in GlnRS15 causes virtually no change in the structure of the complex, and the explanation for its ability to misacylate supF is unclear.     

The angle between the anticodon and aminoacyl acceptor stems of yeast tRNA(Phe) is strongly modulated by magnesium ions

Many tRNAs undergo tertiary folding transitions at temperatures well below the main thermally induced (hyperchromic) transition. Such transitions are essentially isochromic and isoenthalpic and display an absolute requirement for divalent cations; however, the nature of the structural transition is not known for any tRNA. Using a combination of transient electric birefringence (TEB) and gel electrophoretic measurements, we have characterized the influence of magnesium ions on the apparent angle between the anticodon and acceptor stems of a yeast tRNA(Phe) construct. TEB is a particularly sensitive method for quantifying the bends introduced in RNA by various nonhelix elements. In the current instance, the tRNA construct comprises an unmodified tRNA(Phe) molecule in which the anticodon and acceptor stems have been extended by approximately 70 bp to more effectively "report" the interstem angles. Upon the addition of sub-millimolar concentrations of magnesium ions, the tRNA core undergoes a substantial rearrangement in tertiary structure, passing from an open form with an apparent interstem angle of approximately 150 degrees to a conformation with an interstem angle of approximately 70 degrees (200 microM Mg2+). Further addition of magnesium ions results in a minor adjustment of the apparent interstem angle to approximately 80-90 degrees, in line with earlier results. Finally, the magnesium-induced structural transition is essentially isochromic, in agreement with previous observations with native tRNAs. The current results suggest that changes in local divalent ion concentration in the ribosome could profoundly affect the global conformations of tRNAs during the translation cycle.     

Two regions of the Escherichia coli 16S ribosomal RNA are important for decoding stop signals in polypeptide chain termination

Two regions of the 16S rRNA, helix 34, and the aminoacyl site component of the decoding site at the base of helix 44, have been implicated in decoding of translational stop signals during the termination of protein synthesis. Antibiotics specific for these regions have been tested to see how they discriminate the decoding of UAA, UAG, and UGA by the two polypeptide chain release factors (RF-1 and RF-2). Spectinomycin, which interacts with helix 34, stimulated RF-1 dependent binding to the ribosome and termination. It also stimulated UGA dependent RF-2 termination at micromolar concentrations but inhibited UGA dependent RF-2 binding at higher concentrations. Alterations at position C1192 of helix 34, known to confer spectinomycin resistance, reduced the binding of f[3H]Met-tRNA to the peptidyl-tRNA site. They also impaired termination in vitro, with both factors and all three stop codons, although the effect was greater with RF-2 mediated reactions. These alterations had previously been shown to inhibit EF-G mediated translocation. As perturbations in helix 34 effect both termination and elongation reactions, these results indicate that helix 34 is close to the decoding site on the bacterial ribosome. Several antibiotics, hygromycin, neomycin and tetracycline, specific for the aminoacyl site, were shown to inhibit the binding and function of both RFs in termination with all three stop codons in vitro. These studies indicate that decoding of all stop signals is likely to occur at a similar site on the ribosome to the decoding of sense codons, the aminoacyl site, and are consistent with a location for helix 34 near this site.     

Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA6Leu (Y. Komine et al., J. Mol. Biol. 212:579-598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O2-methyluridine) as its anticodon, tRNA6Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6Leu. These tRNA6Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4Leu restored the growth of group I mutants at 42 degrees C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4Leu poorly recognizes the UUG codon at 42 degreesC and that the wild-type tRNA6Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6Leu remains to be investigated.     

Binding of a fragment of yeast phenylalanyl tRNA containing an anticodon loop with 30S and 70S ribosomes from Escherichia coli. The role of guanosine-42 in this interaction

Poly(U)-dependent binding of isolated yeast tRNA(Phe) anticodon hairpin (15-nucleotide-long, corresponding to nucleotides 28-42 within the tRNA) and several its derivatives to the P site of Escherichia coli 30S and 70S ribosomes was studied quantitatively. The affinity for the hairpin binding to 70S ribosomes was shown to be only 30-fold weaker than that for the binding of total tRNA(Phe). Within the anticodon hairpin, removal of the 3'-terminal nucleotide corresponding to guanosine-42 in tRNA(Phe) decreases the association constant for the anticodon arm-ribosome interaction 15-fold. Replacement of this guanosine with other nucleosides does not affect the affinity, regardless of involvement in the hairpin secondary structure. These data indicate that G-42 affects the anticodon arm affinity most likely by forming a direct contact with the ribosome. One can assume that this nucleotide within intact tRNA also forms a contact with the P site. Since the 3'-terminal ribose modifications (oxidation, oxidation and reduction) as well as the presence or absence of the 3'-terminal phosphate does not affect the affinity of the anticodon arm fragment, the latter is obviously involved in the interaction through 3'-terminal nucleotide base groups which does not take part in base pairing.     

A novel ribosome-associated protein is important for efficient translation in Escherichia coli

Previously, we showed that strains which have been deleted for the 21K gene (hereafter called yfjA), of the trmD operon, encoding a 21-kDa protein (21K protein) have an approximately fivefold-reduced growth rate in rich medium. Here we show that such mutants show an up to sevenfold reduced growth rate in minimal medium, a twofold-lower cell yield-to-carbon source concentration ratio, and a reduced polypeptide chain growth rate of beta-galactosidase. Suppressor mutations that increased the growth rate and translational efficiency of a delta yfjA mutant were localized to the 3' part of rpsM, encoding ribosomal protein S13. The 21K protein was shown to have affinity for free 30S ribosomal subunits but not for 70S ribosomes. Further, the 21K protein seems to contain a KH domain and a KOW motif, both suggested to be involved in binding of RNA. These findings suggest that the 21K protein is essential for a proper function of the ribosome and is involved in the maturation of the ribosomal 30S subunits or in translation initiation.