Nucleotides that contribute to the identity of Escherichia coli tRNA(Phe)

A series of sequence variants of amber suppressor genes of tRNA(Phe) were synthesized in vitro and cloned in Escherichia coli to examine the contributions of individual nucleotides to identity for amino acid acceptance. Three different but complementary types of tRNA variants were constructed. The first involved the substitution of base-pairs on the cloverleaf stem regions of the E. coli tRNA(Phe). The second type of variant involved total gene synthesis based on wild-type tRNA(Phe) sequences found in Bacillus subtilis and in Halobacterium volcanii. In the third type of variant, the identity of E. coli tRNALys was changed to that of tRNA(Phe). The nucleotides which are important for tRNA(Phe) identity in E. coli are located on the corner of the L-shaped tRNA molecule, where the dihydrouridine loop interacts with the T loop, and extend to the interior opening of the anticodon stem and the adjoining variable loop. The nucleotide sequence on the dihydrouridine stem region, which joins the corner and stem regions, was not successfully studied though it may contribute to tRNA(Phe) identity. The fourth nucleotide from the 3' end of tRNA(Phe) has some importance for identity.     

Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene.

We describe the cloning and the DNA sequence of an amber suppressor allele of the Escherichia coli leuX (supP) gene. The suppressor allele codes for a tRNA with anticodon CUA, presumably derived by a single base change from a CAA anticodon. The mature coding sequence of the leuX gene is preceded by a putative Pribnow box sequence (TATAAT) and followed by a termination signal. The sequence of the leuX-coded tRNA is compared with the sequences of the four remaining tRNALeu isoacceptors of E. coli and with two tRNALeu species from bacteriophage T4 and T5. The conserved nucleotides in these seven tRNAs recognized by E. coli leucyl-tRNA synthetase are located mainly in the aminoacyl stem and in the D-stem/loop region.     

Importance of the G27-A43 mismatch at the anticodon stem of Escherichia coli tRNA(Thr2)

The tRNA(Thr2) isoacceptor of E. coli has a G-A mismatch at positions 27-43. When the anticodon of this tRNA was converted to an amber anticodon (CUA), this tRNA showed suppressor activity in E. coli. Moreover, introduction of the base pair (G-C or U-A) at positions 27-43 of this suppressor tRNA reduced its suppressor activity. These results indicate that the G27-A43 mismatch is necessary for full function of tRNA(Thr2).     

Plant nonsense suppressor tRNA(Tyr) genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs.

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.     

Synthetase competition and tRNA context determine the in vivo identify of tRNA discriminator mutants.

The discriminator nucleotide (position 73) in tRNA has long been thought to play a role in tRNA identity as it is the only variable single-stranded nucleotide that is found near the site of aminoacylation. For this reason, a complete mutagenic analysis of the discriminator in three Escherichia coli amber suppressor tRNA backgrounds was undertaken; supE and supE-G1C72 glutamine tRNAs, gluA glutamate tRNA and supF tyrosine tRNA. The effect of mutation of the discriminator base on the identity of these tRNAs in vivo was assayed by N-terminal protein sequencing of E. coli dihydrofolate reductase, which is the product of suppression by the mutated amber suppressors, and confirmed by amino acid specific suppression experiments. In addition, suppressor efficiency assays were used to estimate the efficiency of aminoacylation in vivo. Our results indicate that the supE glutamine tRNA context can tolerate multiple mutations (including mutation of the discriminator and first base-pair) and still remain predominantly glutamine-accepting. Discriminator mutants of gluA glutamate tRNA exhibit increased and altered specificity probably due to the reduced ability of other synthetases to compete with glutamyl-tRNA synthetase. In the course of these experiments, a glutamate-specific mutant amber suppressor, gluA-A73, was created. Finally, in the case of supF tyrosine tRNA, the discriminator is an important identity element with partial to complete loss of tyrosine specificity resulting from mutation at this position. It is clear from these experiments that it may not be possible to assign a specific role in tRNA identity to the discriminator. The identity of a tRNA in vivo is determined by competition among aminoacyl-tRNA synthetases, which is in turn modulated by the nucleotide substitution as well as the tRNA context.     

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.     

Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that are highly specific for their tRNA substrates. Here, we describe the expansion of a class IIb aaRS-tRNA specificity by a genetic selection that involves the use of a modified tRNA displaying an amber anticodon and the argE(amber) and lacZ(amber) reporters. The study was performed on Escherichia coli aspartyl-tRNA synthetase (AspRS) and amber tRNA(Asp). Nine AspRS mutants able to charge the amber tRNA(Asp) and to suppress the reporter genes were selected from a randomly mutated library. All the mutants exhibited a new amber tRNA(Asp) specificity in addition to the initial native tRNA(Asp). Six mutations were found in the anticodon-binding site located in the N-terminal OB-fold. The strongest suppressor was a mutation of residue Glu-93 that contacts specifically the anticodon nucleotide 34 in the crystal structure. The other mutations in the OB-fold were found at close distance from the anticodon in the so-called loop L45 and strand S1. They concern residues that do not contact tRNA(Asp) in the native complex. In addition, this study shows that suppressors can carry mutations located far from the anticodon-binding site. One such mutation was found in the synthetase hinge-module where it increases the tRNA(Asp)-charging rate, and two other mutations were found in the prokaryotic-specific insertion domain and the catalytic core. These mutants seem to act by indirect effects on the tRNA acceptor stem binding and on the conformation of the active site of the enzyme. Altogether, these data suggest the existence of various ways for modifying the mechanism of tRNA discrimination.     

Selection of tRNA(Asp) amber suppressor mutants having alanine, arginine, glutamine, and lysine identity.

Elements that confer identity to a tRNA in the cellular environment, where all aminoacyl-tRNA synthetases are competing for substrates, may be delineated by in vivo experiments using suppressor tRNAs. Here we describe the selection of active Escherichia coli tRNAAsp amber mutants and analyze their identity. Starting from a library containing randomly mutated tRNA(CUA)Asp genes, we isolated four amber suppressors presenting either lysine, alanine, or glutamine activity. Two of them, presenting mainly alanine or lysine activity, were further submitted to a second round of mutagenesis selection in order to improve their efficiency of suppression. Eleven suppressors were isolated, each containing two or three mutations. Ten presented identities of the two parental mutants, whereas one had switched from lysine to arginine identity. Analysis of the different mutants revealed (or confirmed for some nucleotides) their role as positive and/or negative determinants in AlaRS, LysRS, and ArgRS recognition. More generally, it appears that tRNAAsp presents identity characteristics closely related to those of tRNALys, as well as a structural basis for acquiring alanine or arginine identity upon moderate mutational changes; these consist of addition or suppression of the corresponding positive or negative determinants, as well as tertiary interactions. Failure to isolate aspartic acid-inserting suppressors is probably due to elimination of the important G34 identity element and its replacement by an antideterminant when changing the anticodon of the tRNAAsp to the CUA triplet.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.     

Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.     

Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation.

The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu). Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.     

In vitro conversion of a methionine to a glutamine-acceptor tRNA

A derivative of Escherichia coli tRNAfMet containing an altered anticodon sequence, CUA, has been enzymatically synthesized in vitro. The variant tRNA was prepared by excision of the normal anticodon, CAU, in a limited digestion of intact tRNAfMet with RNase A, followed by insertion of the CUA sequence into the anticodon loop with T4 RNA ligase and polynucleotide kinase. The altered methionine tRNA showed a large enhancement in the rate of aminoacylation by glutaminyl-tRNA synthetase and a large decrease in the rate of aminoacylation by methionyl-tRNA synthetase. Measurement of kinetic parameters for the charging reaction by the cognate and noncognate enzymes revealed that the modified tRNA is a better acceptor for glutamine than for methionine. The rate of mischarging is similar to that previously reported for a tryptophan amber suppressor tRNA containing the anticodon CUA, su+7 tRNATrp, which is aminoacylated with glutamine both in vivo and in vitro [Yaniv, M., Folk, W. R., Berg, P., & Soll, L. (1974) J. Mol. Biol. 86, 245-260; Yarus, M., Knowlton, R. E., & Soll, L. (1977) in Nucleic Acid-Protein Recognition (Vogel, H., Ed.) pp 391-408, Academic Press, New York]. The present results provide additional evidence that the specificity of aminoacylation by glutaminyl-tRNA synthetase is sensitive to small changes in the nucleotide sequence of noncognate tRNAs and that uridine in the middle position of the anticodon is involved in the recognition of tRNA substrates by this enzyme.     

Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base,amber, and opal suppression

Recently, it has been shown that an amber suppressor tRNA/aminoacyl-tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jannaschii can be used to genetically encode unnatural amino acids in response to the amber nonsense codon, TAG. However, we have been unable to modify this pair to decode either the opal nonsense codon, TGA, or the four-base codon, AGGA, limiting us to a 21 amino acid code. To overcome this limitation, we have adapted a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and leucyl tRNA derived from Halobacterium sp. NRC-1 as an orthogonal tRNA-synthetase pair in Escherichia coli to decode amber (TAG), opal (TGA), and four-base (AGGA) codons. To improve the efficiency and selectivity of the suppressor tRNA, extensive mutagenesis was performed on the anticodon loop and acceptor stem. The two most significant criteria required for an efficient amber orthogonal suppressor tRNA are a CU(X)XXXAA anticodon loop and the lack of noncanonical or mismatched base pairs in the stem regions. These changes afford only weak suppression of TGA and AGGA. However, this information together with an analysis of sequence similarity of multiple native archaeal tRNA sequences led to efficient, orthogonal suppressors of opal codons and the four-base codon, AGGA. Ultimately, it should be possible to use these additional orthogonal pairs to genetically incorporate multiple unnatural amino acids into proteins.