The anticodon and discriminator base are major determinants of cysteine tRNA identity in vivo.

Mutants of the Escherichia coli initiator tRNA (tRNA(fMet)) have been used to examine the role of the anticodon and discriminator base in in vivo aminoacylation of tRNAs by cysteinyl-tRNA synthetase. Substitution of the methionine anticodon CAU with the cysteine anticodon GCA was found to allow initiation of protein synthesis by the mutant tRNA from a complementary initiation codon in a reporter protein. Sequencing of the protein revealed that cysteine comprised about half of the amino acid at the N terminus. An additional mutation, converting the discriminator base of tRNA(GCAfMet) from A73 to the base present in tRNA(Cys) (U73), resulted in a 6-fold increase in the amount of protein produced and insertion of greater than or equal to 90% cysteine in response to the complementary initiation codon. Substitution of C73 or G73 at the discriminator position led to insertion of little or no cysteine, indicating the importance of U73 for recognition of the tRNA by cysteinyl-tRNA synthetase. Single base changes in the anticodon of tRNA(GCAfMet) containing U73 from GCA to UCA, GUA, GCC, and GCG (changes underlined) eliminated or dramatically reduced cysteine insertion by the mutant initiator tRNA indicating that all three cysteine anticodon bases are essential for specific aminoacylation of the tRNA with cysteine in vivo.     

E. coli initiator tRNA analogs with different nucleotides in the discriminator base position.

The effect of base changes at the fourth position from the 3'-terminus of Escherichia coli initiator tRNAMet has been studied to test the 'discriminator hypothesis' which proposed that the nucleotide in this position might have a role in the specificity of the aminoacylation reaction. E. coli initiator tRNA lacking the 3'-terminal tetranucleotide was prepared by partial digestion with S1 nuclease. To construct tRNA analogs with different bases in the fourth position this truncated tRNA was joined by RNA ligase to each of four chemically synthesized 2',3'-ethoxy-methylidene tetranucleotides pACCA(em), pCCCA(em), pGCCA(em), and pUCCA(em). In vitro aminoacylation studies showed that all four molecules accepted methionine, albeit with different Vmax values.     

Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates.

We show here that yeast tRNA ligase protein is essential in the cell and participates in joining together tRNA half-molecules resulting from excision of the intron by the splicing endonuclease. A haploid yeast strain carrying a chromosomal deletion of the ligase gene is viable only if ligase protein can be supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase gene is repressed, cells eventually die and accumulate endonuclease cut but unligated half-molecules and intervening sequences. Half-molecules that accumulate appear to be fully end-processed. Two temperature-sensitive ligase mutant strains have been isolated; these strains accumulate a similar set of unligated half-molecules at the nonpermissive temperature.     

Polymerization of the Tryptophanyl tRNA Synthetase Complex with Tryptophan

To understand the mechanism of mutual recognition between aminoacyl tRNA synthetases (EC 6.1.1) and tRNAs, it is important to obtain more information about the structure of these enzymes. X-ray crystallography offers one approach, but although several aminoacyl tRNA synthetases have been isolated in very pure form¹ and crystallization of tryptophanyl² and seryl³ tRNA synthetases has been reported, no crystals were available that were suitable for structural analysis. Only very recently have Rymo and Lagerkvist⁴ described the formation of crystals of yeast lysyl tRNA synthetase which are apparently suitable for further X-ray analysis. Here we deal with the phenomenon of polymerization of the tryptophanyl tRNA synthetase complex with tryptophan in the form of rod-like particles and the subsequent formation of para-crystalline aggregates.     

Threonyl-tRNA synthetase of archaea: importance of the discriminator base in the aminoacylation of threonine tRNA.

To investigate the contribution of the discriminator base of archaeal tRNA(Thr) in aminoacylation by threonyl-tRNA synthetase (ThrRS), cross-species aminoacylation between Escherichia coli and Haloferax volcanii, halophilic archaea, was studied. It was found that E. coli ThrRS threonylated the H. volcanii tRNA(Thr) but that E. coli threonine tRNA was not aminoacylated by H. volcanii ThrRS. Results of a threonylation experiment using in vitro mutants of E. coli threonine tRNA showed that only the mutant tRNA(Thr) having U73 was threonylated by H. volcanii ThrRS. These findings indicate that the discriminator base U73 of H. volcanii tRNA(Thr) is a strong determinant for the recognition by ThrRS.     

Biosynthesis of tRNA in histidine regulatory mutants of Salmonella typhimurium.

HisU mutants of Salmonella typhimurium are depressed in the histidine operon since they have lower intracellular concentration of histidyl-tRNAHis. In this paper we present evidences showing that a strain carrying a hisU mutation (hisUl206) is altered in a nucleolytic enzyme involved in tRNA maturation process. The analysis of several hisU mutants indicates that hisU region of bacterial genome may account for more than one function involved in tRNA biosynthesis.     

Molecular Evolution of Aminoacyl tRNA Synthetase Proteins in the Early History of Life

Aminoacyl-tRNA synthetases (aaRS) consist of several families of functionally conserved proteins essential for translation and protein synthesis. Like nearly all components of the translation machinery, most aaRS families are universally distributed across cellular life, being inherited from the time of the Last Universal Common Ancestor (LUCA). However, unlike the rest of the translation machinery, aaRS have undergone numerous ancient horizontal gene transfers, with several independent events detected between domains, and some possibly involving lineages diverging before the time of LUCA. These transfers reveal the complexity of molecular evolution at this early time, and the chimeric nature of genomes within cells that gave rise to the major domains. Additionally, given the role of these protein families in defining the amino acids used for protein synthesis, sequence reconstruction of their pre-LUCA ancestors can reveal the evolutionary processes at work in the origin of the genetic code. In particular, sequence reconstructions of the paralog ancestors of isoleucyl- and valyl- RS provide strong empirical evidence that at least for this divergence, the genetic code did not co-evolve with the aaRSs; rather, both amino acids were already part of the genetic code before their cognate aaRSs diverged from their common ancestor. The implications of this observation for the early evolution of RNA-directed protein biosynthesis are discussed.     

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants.     

The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNA^Ile organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNA^Ile affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNA^Ile synthesis under cellular conditions. Finally, the extent to which tRNA^Ile modulates activation and pre-transfer editing is independent of the intactness of its 3′-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3′-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.     

大肠杆菌精氨酰tRNA合成酶基因的诱导高表达

在高表达大肠杆菌精氨酰ｔＲＮＡ合成酶基因５５０倍的基础上,将ａｒｇ２的编码起始位点经基因突点突变导入ＮｃｏＩ限制性内切酶的位点后,重组到受异丙基硫代－β－Ｄ半乳糖苷诱导的ｐＴｒ９９Ｂ质粒上,使ａｒｇＳ比受体菌表达高近２０００倍。通过一步ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析则可得到ＳＤＳ－ＰＡＧＥ一条带的ＡｒｇＲＳ,比活为１５０００ｕ／ｍｇ,与文献相同。     

Construction of region-specific partial duplication mutants (merodiploid mutants) to identify the regulatory gene for the glucan-binding protein C gene in vivo in Streptococcus mutans

The gbpC gene encoding the glucan-binding protein C which is involved in dextran (glucan)-dependent aggregation (ddag) of Streptococcus mutans has been identified by random mutagenesis. We analyzed ddag(-) mutants containing the intact gbpC gene and found that these mutants possessed a large and characteristic duplication of a region of the chromosome which was responsible for the phenotype. Based upon characterization of these duplications, we developed a strategy to introduce a duplication into any specific region of the chromosome of these organisms. The 690-bp gene responsible for the ddag(-) phenotype was identified within a 60-kb region by observing ddag (positive or negative) phenotypes of successively constructed specific duplication mutants.     

Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.

We describe the use of a gel electrophoretic method for measuring the levels of aminoacylation in vivo of mutant Escherichia coli initiator tRNAs, which are substrates for E. coli glutaminyl-tRNA synthetase (GlnRS) due to an anticodon sequence change. Using this method, we have compared the effects of introducing further mutations in the acceptor stem, at base pairs 1:72, 2:71, and 3:70 and discriminator base 73, on the recognition of these tRNAs by E. coli GlnRS in vitro and in vivo. The effects of the acceptor stem mutations on the kinetic parameters for aminoacylation of the mutant tRNAs in vitro are consistent with interactions seen between this region of tRNA and GlnRS in the crystal structure of tRNA(Gln). GlnRS complex. Except for one mutant, the observed levels of aminoacylation of the mutant tRNAs in vivo agree with those expected on the basis of the kinetic parameters obtained in vitro. We have also measured the relative amounts of aminoacyl-tRNAs for the various mutants and their activities in suppression of an amber codon in vivo. We find that there is, in general, a good correlation between the relative amounts of aminoacyl-tRNAs and their activities in suppression.