Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria.

Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn)and tRNA(Lys)were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys)corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met)corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system. tRNA(Ser)GCU corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or G at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system.     

Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1(Ser) gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1(Ser). Several genes containing UCA codons are normally expressed at 42 degrees C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1(Ser). In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.     

Codon reading by tRNAAla with modified uridine in the wobble position

tRNAs reading four-codon families often have a modified uridine, cmo(5)U(34), at the wobble position of the anticodon. Here, we examine the effects on the decoding mechanism of a cmo(5)U modification in tRNA(1B)(Ala), anticodon C(36)G(35)cmo(5)U(34). tRNA(1B)(Ala) reads its cognate codons in a manner that is very similar to that of tRNA(Phe). As Ala codons are GC rich and Phe codons AU rich, this similarity suggests a uniform decoding mechanism that is independent of the GC content of the codon-anticodon duplex or the identity of the tRNA. The presence of cmo(5)U at the wobble position of tRNA(1B)(Ala) permits fairly efficient reading of non-Watson-Crick and nonwobble bases in the third codon position, e.g., the GCC codon. The ribosome accepts the C-cmo(5)U pair as an almost-correct base pair, unlike third-position mismatches, which lead to the incorporation of incorrect amino acids and are efficiently rejected.     

Third position base changes in codons 5' and 3' adjacent UGA codons affect UGA suppression in vivo

The base sequence around nonsense codons affects the efficiency of nonsense codon suppression. Published data, comparing different nonsense sites in a mRNA, implicate the two bases downstream of the nonsense codon as major determinants of suppression efficiency. However, the results we report here indicate that the nature of the contiguous upstream codon can also affect nonsense suppression, as can the third (wobble) base of the contiguous downstream codon. These conclusions are drawn from experiments in which the two Ser codons UCU233 and UCG235 in a nonsense mutant form (UGA234) of the trpA gene in Escherichia coli have been replaced with other Ser codons by site-directed mutagenesis. Suppression of these trpA mutants has been studied in the presence of a UGA nonsense suppressor derived from glyT. We speculate that the non-site-specific effects of the two adjacent downstream bases may be largely at the level of the termination process, whereas more site-specific or codon-specific effects may operate primarily on the activity of the suppressor tRNA.     

Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro.

Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.     

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.     

Accurate translation of the genetic code depends on tRNA modified nucleosides

Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3' to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U(34) to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s(2)U(34)), 5-methylaminomethyluridine at position 34 (mnm(5)U(34)), and 6-threonylcarbamoyladenosine at position 37 (t(6)A(37)) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA(UUU)(Lys) at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.     

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)     

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.     

Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria

The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.     

The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo

In Salmonella enterica serovar Typhimurium five of the eight family codon boxes are decoded by a tRNA having the modified nucleoside uridine-5-oxyacetic acid (cmo5U) as a wobble nucleoside present in position 34 of the tRNA. In the proline family codon box, one (tRNAProcmo5UGG) of the three tRNAs that reads the four proline codons has cmo5U34. According to theoretical predictions and several results obtained in vitro, cmo5U34 should base pair with A, G, and U in the third position of the codon but not with C. To analyze the function of cmo5U34 in tRNAProcmo5UGG in vivo, we first identified two genes (cmoA and cmoB) involved in the synthesis of cmo5U34. The null mutation cmoB2 results in tRNA having 5-hydroxyuridine (ho5U34) instead of cmo5U34, whereas the null mutation cmoA1 results in the accumulation of 5-methoxyuridine (mo5U34) and ho5U34 in tRNA. The results suggest that the synthesis of cmo5U34 occurs as follows: U34 -->(?) ho5U -->(CmoB) mo5U -->(CmoA?) cmo5U. We introduced the cmoA1 or the cmoB2 null mutations into a strain that only had tRNAProcmo5UGG and thus lacked the other two proline-specific tRNAs normally present in the cell. From analysis of growth rates of various strains and of the frequency of +1 frameshifting at a CCC-U site we conclude: (1) unexpectedly, tRNAProcmo5UGG is able to read all four proline codons; (2) the presence of ho5U34 instead of cmo5U34 in this tRNA reduces the efficiency with which it reads all four codons; and (3) the fully modified nucleoside is especially important for reading proline codons ending with U or C.     

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.     

Functional importance of RNA interactions in selection of translation initiation codons

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA–small ribosomal subunit–initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis -acting mRNA elements and trans -acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.     

Wobble modification defect in tRNA disturbs codon-anticodon interaction in a mitochondrial disease

We previously showed that in mitochondrial tRNA(Lys) with an A8344G mutation responsible for myoclonus epilepsy associated with ragged-red fibers (MERRF), a subgroup of mitochondrial encephalomyopathic diseases, the normally modified wobble base (a 2-thiouridine derivative) remains unmodified. Since wobble base modifications are essential for translational efficiency and accuracy, we used mitochondrial components to estimate the translational activity in vitro of purified tRNA(Lys) carrying the mutation and found no mistranslation of non-cognate codons by the mutant tRNA, but almost complete loss of translational activity for cognate codons. This defective translation was not explained by a decline in aminoacylation or lowered affinity toward elongation factor Tu. However, when direct interaction of the codon with the mutant tRNA(Lys) defective anticodon was examined by ribosomal binding analysis, the wild-type but not the mutant tRNA(Lys) bound to an mRNA- ribosome complex. We therefore concluded that the anticodon base modification defect, which is forced by the pathogenic point mutation, disturbs codon- anticodon pairing in the mutant tRNA(Lys), leading to a severe reduction in mitochondrial translation that eventually could result in the onset of MERRF.