The Escherichia coli argU10(Ts) phenotype is caused by a reduction in the cellular level of the argU tRNA for the rare codons AGA and AGG

The Escherichia coli argU10(Ts) mutation in the argU gene, encoding the minor tRNA(Arg) species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30 degrees C. In the present study, we first showed that the codon selectivity and the arginine-accepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation. An in vitro analysis of the effects of the argU10(Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10(Ts) mutant tRNA is impaired at 30 and 43 degrees C, and this defect is more serious at the higher temperature. The arginine acceptance is also impaired significantly but to similar extents at the two temperatures. An in vivo analysis of aminoacylation levels showed that 30% of the argU10(Ts) tRNA molecules in the mutant cells are actually deacylated at 30 degrees C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated. Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA. At 43 degrees C, the cellular level of the argU10(Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes. We concluded that the phenotypic properties of the argU10(Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase.     

A minor arginine tRNA mutant limits translation preferentially of a protein dependent on the cognate codon.

The Escherichia coli argU gene encodes a rare arginine tRNA (anticodon UCU) that translates the similarly rare AGA codon. The argU10(Ts) mutation is a transition that changes the first nucleotide of the mature tRNA from G to A, presumably destabilizing the acceptor stem. This mutation, when present in haploid condition in the chromosome, reduces the growth rate at 30 degrees C and results in cessation of growth after 60 to 90 min at 43 degrees C. The mutation also preferentially limits (compared with total protein synthesis) translation of an induced gene that depends on five AGA codons, i.e., the lambda cI repressor gene. Translation of another inducible protein, beta-galactosidase, which does not involve AGA codons, was inhibited to a much lesser extent. The chromosomal argU(Ts) mutation also confers the Pin phenotype, that is, loss of ability of the host, as a P2 lysogen, to inhibit growth of bacteriophage lambda, probably the result of reduced translation of the P2 old gene, which contains five AGA codons (E. Hagg?rd-Ljungquist, V. Barreiro, R. Calendar, D. M. Kurnit, and H. Cheng, Gene 85:25-33, 1989).     

An extra tRNAGly(U*CU) found in ascidian mitochondria responsible for decoding non-universal codons AGA/AGG as glycine.

Amino acid assignments of metazoan mitochondrial codons AGA/AGG are known to vary among animal species; arginine in Cnidaria, serine in invertebrates and stop in vertebrates. We recently found that in the mitochondria of the ascidian Halocynthia roretzi these codons are exceptionally used for glycine, and postulated that they are probably decoded by a tRNA(UCU). In order to verify this notion unambig-uously, we determined the complete RNA sequence of the mitochondrial tRNA(UCU) presumed to decode codons AGA/AGG in the ascidian mitochondria, and found it to have an unidentified U derivative at the anticodon first position. We then identified the amino acids attached to the tRNA(U*CU), as well as to the conventional tRNAGly(UCC) with an unmodified U34, in vivo. The results clearly demonstrated that glycine was attached to both tRNAs. Since no other tRNA capable of decoding codons AGA/AGG has been found in the mitochondrial genome, it is most probable that this tRNA(U*CU) does actually translate codons AGA/AGG as glycine in vivo. Sequencing of tRNASer(GCU), which is thought to recognize only codons AGU/AGC, revealed that it has an unmodified guanosine at position 34, as is the case with vertebrate mitochondrial tRNASer(GCU) for codons AGA/AGG. It was thus concluded that in the ascidian, codons AGU/AGC are read as serine by tRNASer(GCU), whereas AGA/AGG are read as glycine by an extra tRNAGly(U*CU). The possible origin of this unorthodox genetic code is discussed.     

In vitro codon-reading specificities of unmodified tRNA molecules with different anticodons on the sequence background of Escherichia coli tRNASer

The codon-reading properties of wobble-position variants of the unmodified form of Escherichia coli tRNASer1 (the UGA anticodon) were measured in a cell-free translation system. Two variants, with the AGA and CGA anticodons, each exclusively read a single codon, UCU and UCG, respectively. The only case of efficient wobbling occurred with the variant with the GGA anticodon, which reads the UCU codon in addition to the UCC codon. Surprisingly, this wobble reading is more efficient than the Watson-Crick reading by the variant with the AGA anticodon. Furthermore, we prepared tRNA variants with AA, UC, and CU, instead of GA, in the second and third positions and measured their relative efficiencies in the reading of codons starting with UU, GA, and AG, respectively. The specificity concerning the wobble position is essentially the same as that in the case of the codons starting with UC.     

tRNA-assisted overproduction of eukaryotic ribosomal proteins

Structural studies of eukaryotic ribosomes are complicated by the tendency of their constituent proteins to be expressed at very low levels in Escherichia coli. We find that this is mainly due to their exceptionally high content of AGA/AGG arginine codons, which are poorly utilized by the bacterial translational machinery. In fact, we could overcome this limitation by the combined use of a T7 RNA polymerase expression vector and a plasmid carrying the E. coli gene argU, which encodes the minor tRNA(Arg) species that reads AGA/AGG codons. In this system, five cytoplasmic ribosomal proteins from three different eukaryotic lineages (Saccharomyces cerevisiae S8, L13, and L14; Arabidopsis thaliana L13; and Homo sapiens L7) could be overexpressed to up to 50% of total bacterial protein and were purified to homogeneity in tens of milligrams amounts. The purification procedure simply involved metal affinity chromatography followed, in some cases, by an additional heparin chromatography step. Recombinant polypeptides bound RNA with high affinity (K(d) between 50 and 300 nM). This novel overexpression/purification strategy will allow the production of high amounts of most eukaryotic ribosomal proteins in a form suitable for structural and functional studies. Coupled with recently completed and ongoing whole-genome sequencing projects, it will facilitate the molecular characterization of the eukaryotic ribosome.     

Expression and characterization of the HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus

The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA(AGA,AGG), the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA(AGA,AGG) resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K(i) for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.     

Evolution of the mitochondrial genetic code I. Origin of AGR serine and stop codons in metazoan mitochondria

Summary AGA and AGG (AGR) are arginine codons in the universal genetic code. These codons are read as serine or are used as stop codons in metazoan mitochondria. The arginine residues coded by AGR in yeast orTrypanosoma are coded by arginine CGN throughout metazoan mitochondria. AGR serine sites in metazoan mitochondria are occupied mainly in corresponding sites in yeast orTrypanosoma mitochondria by UCN serine, AGY serine, or codons for amino acids other than serine or arginine. Based on these observations, we propose the following evolutionary events. AGR codons became unassigned because of deletion of tRNA Arg (UCU) and elimination of AGR codons by conversion to CGN arginine codons. Upon acquisition by serine tRNA of pairing ability with AGR codons, some codons for amino acids other than arginine mutated to AGR, and were caputed by anticodon GCU in serine tRNA. During vertebrate mitochondrial evolution, AGR stop codons presumably were created from UAG stop by deletion of the first nucleotide U and by use of R as the third nucleotide that had existed next to the ancestral UAG stop.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

A reduced level of charged tRNAArgmnm5UCU triggers the wild-type peptidyl-tRNA to frameshift

Frameshift mutations can be suppressed by a variety of differently acting external suppressors. The +1 frameshift mutation hisC3072, which has an extra G in a run of Gs, is corrected by the external suppressor mutation sufF44. We have shown that sufF44 and five additional allelic suppressor mutations are located in the gene argU coding for the minor tRNAArgmnm5UCU and alter the secondary and/or tertiary structure of this tRNA. The C61U, G53A, and C32U mutations influence the stability, whereas the C56U, C61U, G53A, and G39A mutations decrease the arginylation of tRNAArgmnm5UCU. The T-10C mutant has a base substitution in the -10 consensus sequence of the argU promoter that reduces threefold the synthesis of tRNAArgmnm5UCU . The lower amount of tRNAArgmnm5UCU or impaired arginylation, either independently or in conjunction, results in inefficient reading of the cognate AGA codon that, in turn, induces frameshifts. According to the sequence of the peptide produced from the suppressed -GGG-GAA-AGA- frameshift site, the frameshifting tRNA in the argU mutants is tRNAGlumnm5s2UUC, which decodes the GAA codon located upstream of the AGA arginine codon, and not the mutated tRNAArgmnm5UCU. We propose that an inefficient decoding of the AGA codon by a defective tRNAArgmnm5UCU stalls the ribosome at the A-site codon allowing the wild-type form of peptidyl-tRNAGlumnm5s2UUC to slip forward 1 nucleotide and thereby re-establish the ribosome in the 0-frame. Similar frame-shifting events could be the main cause of various phenotypes associated with environmental or genetically induced changes in the levels of aminoacylated tRNA.     

Unusual genetic codes and a novel gene structure for tRNA(AGYSer) in starfish mitochondrial DNA

The nucleotide sequence of a 3849-bp fragment of starfish mitochondrial genome was determined. The genes for NADH dehydrogenase subunits 3, 4, 5, and COIII, and three kinds of (tRNA(UCNSer), tRNA(His), and tRNA(AGYSer) were identified by comparing with the genes of other animal mitochondria so far elucidated. The gene arrangement of starfish mitochondrial genome was different from those of vertebrate and insect mitochondrial genomes. Comparison of the protein-encoding nucleotide sequences of starfish mitochondria with those of other animal mitochondria suggested a unique genetic code in starfish mitochondrial genome; both AGA and AGG (arginine in the universal code) code for serine, AUA (isoleucine in the universal code but methionine in most mitochondrial systems) for isoleucine, and AAA (lysine) for asparagine. It was also inferred that these AGA and AGG codons are decoded by serine tRNA(AGYSer) originally corresponding to AGC and AGU codons. This situation is similar to the case of Drosophila mitochondrial genome. Variations in the use of AGA and AGG codons were discussed on the basis of the evolution of animals and decoding capacity of various tRNA(AGYSer) species possessing different sizes of the dihydrouridine (D) arm.     

A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine

Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA.     

A cluster of six tRNA genes in Drosophila mitochondrial DNA that includes a gene for an unusual tRNAserAGY.

Genes for URF3, tRNAala, tRNAarg, tRNAasn, tRNAserAGY, tRNAglu, tRNAphe, and the carboxyl terminal segment of the URF5 gene have been identified within a sequenced segment of the mtDNA molecule of Drosophila yakuba. The genes occur in the order given. The URF5 and tRNAphe genes are transcribed in the same direction as replication while the URF3 and remaining five tRNA genes are transcribed in the opposite direction. Considerable differences exist in the relative arrangement of these genes in D. yakuba and mammalian mtDNA molecules. In the tRNAserAGY gene an eleven nucleotide loop, within which secondary structure formation seems unlikely, replaces the dihydrouridine arm, and both the variable loop (six nucleotides) and the T phi C loop (nine nucleotides) are larger than in any other D. yakuba tRNA gene. As available evidence is consistent with AGA codons specifying serine rather than arginine in the Drosophila mitochondrial genetic code, the possibility is considered that the 5'GCU anticodon of the D. yakuba tRNAserAGY gene can recognize AGA as well as AGY codons.     

Escherichia coli tRNA(4)(Arg)(UCU) induces a constrained conformation of the crucial Omega-loop of arginyl-tRNA synthetase

Previous investigations show that tRNA(Arg)-induced conformational changes of arginyl-tRNA synthetase (ArgRS) Omega-loop region (Escherichia coli (E. coli), Ala451-Ala457) may contribute to the productive conformation of the enzyme catalytic core, and E. coli tRNA(2)(Arg)(ICG)-bound and -free conformations of the Omega-loop exchange at an intermediate rate on NMR timescale. Herein, we report that E. coli ArgRS catalyzes tRNA(2)(Arg)(ICG) and tRNA(4)(Arg)(UCU) with similar efficiencies. However, 19F NMR spectroscopy of 4-fluorotryptophan-labeled E. coli ArgRS reveals that the tRNA(4)(Arg)(UCU)-bound and -free conformations of the Omega-loop region interconvert very slowly and the lifetime of bound conformation is much longer than 0.33 ms. Therefore, tRNA(4)(Arg)(UCU) differs from tRNA(2)(Arg)(ICG) in the conformation-exchanging rate of the Omega-loop. Comparative structure model of E. coli ArgRS is presented to rationalize these 19F NMR data. Our 19F NMR and catalytic assay results suggest that the tRNA(Arg)-induced conformational changes of Omega-loop little contribute to the productive conformation of ArgRS catalytic core.     

Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage

We previously cloned a panel of peanut allergens by phage display technology. Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene. To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)(10)-tagged fusion proteins in conventional E. coli BL21(DE3) cells. The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8-10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells. Hence, by using modified BL21(DE3) E. coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E. coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage. IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E. coli hosts. The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)(10)-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells. This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells.     

High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product

We have observed that proteins, such as human tissue-type plasminogen activator, pro-urokinase or gp41 of human immunodeficiency virus, which have a high content of rare codons in their respective genes, are not readily expressed in Escherichia coli. Furthermore induction of these heterologous genes leads to growth inhibition and plasmid instability. Supplementation with tRNA(AGA/AGG(Arg)) by cotransfection with the dnaY gene, which supplies this minor tRNA, resulted in high-level production with greatly improved cell viability and plasmid stability.