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PubMed1992

A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha.

Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available.

Amino Acid Sequence↗
PubMed1995

RNA recognition based on a pair of tertiary hydrogen interaction.

The recognition of transfer RNAs (tRNAs) by aminoacyl tRNA synthetases is a critical step in establishing the fidelity of translation. For E. coli cysteine tRNA synthetase, recognition of tRNA(Cys) and discrimination from all other tRNAs is based on the U73 discriminator base, the GCA anticodon, and a G15:G48 tertiary base pair. While the discriminator base and the anticodon sequence are often used by many synthetases as the determinants for tRNA recognition, the dependence on a tertiary interaction in the cognate tRNA for recognition is unique to the cysteine enzyme. Here the structural basis for recognition at the G15:G48 tertiary interaction of E. coli tRNA(Cys) is explored by structural modeling, chemical modifications, and kinetic analysis. The results established an unusual RNA tertiary interaction that provides a plausible mechanism for recognition by cysteine tRNA synthetase.

Anticodon↗
PubMed1993

Identity of Escherichia coli tRNA(Cys) determined by nucleotides in three regions of tRNA tertiary structure.

The in vivo aminoacylation specificity of tRNA(Cys) was studied with mutants of opal and amber suppressor tRNAs in Escherichia coli. This specificity depends not only on nucleotides in the acceptor end and anticodon of tRNA(Cys), but also on nucleotides in the dihydrouridine stem and loop and the variable loop that interact, forming the complex core of tRNA tertiary structure.

Amino Acid Sequence↗
PubMed2004

Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu.

During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu(mt)). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu(mt). Pro269 reorients the loop containing this residue and shifts the adjoining beta-strand in EF-Tu(mt) compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu(mt). For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu(mt) P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA(Phe) and Cys-tRNA(Cys). They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA(Phe). In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA(Phe).

Amino Acid Sequence↗
PubMed1994

Detection of a conserved arrangement of three tRNA genes in the sunflower mitochondrial genome. Identification, mapping and expression of trnC-trnN-trnY genes.

The genes coding for tRNA-Cys (trnC), tRNA-Asn (trnN) and tRNA-Tyr (trnY) have been sequenced in a region of about 3.0 kb of the sunflower mitochondrial DNA. The trnC and trnY are genuine mitochondrial genes, while the trnN gene has a chloroplast origin. Despite their heterologous origin the three genes are transcribed. Their arrangement is the first detected in a highly conserved form in a specific group of advanced dicots.

Base Sequence↗
PubMed2005

Breaking the stereo barrier of amino acid attachment to tRNA by a single nucleotide.

Aminoacyl-tRNA synthetases are responsible for attaching amino acid residues to the tRNA 3'-end. The two classes of synthetases approach tRNA as mirror images, with opposite but symmetrical stereochemistries that allow the class I enzymes to attach amino acid residues to the 2'-hydroxyl group of the terminal ribose, whereas, the class II enzymes attach amino acid residues to the 3'-hydroxyl group. However, we show here that the attachment of cysteine to tRNA(Cys) by the class I cysteinyl-tRNA synthetase (CysRS) is flexible; the enzyme is capable of using either the 2' or 3'-hydroxyl group as the attachment site. The molecular basis for this flexibility was investigated. Introduction of the nucleotide U73 of tRNA(Cys) into tRNA(Val) was found to confer the flexibility. While valylation of the wild-type tRNA(Val) by the class I ValRS was strictly dependent on the terminal 2'-hydroxyl group, that of the U73 mutant of tRNA(Val) occurred at either the 2' or 3'-hydroxyl group. Thus, the single nucleotide U73 of tRNA has the ability to break the stereo barrier of amino acid attachment to tRNA, by mobilizing the 2' and 3'-hydroxyl groups of A76 in flexible geometry with respect to the tRNA acceptor stem.

Amino Acids↗
PubMed1989

Duplication of the tRNA(MMet) and tRNA(Cys) genes and of fragments of a gene encoding a subunit of the NADH dehydrogenase complex in Neurospora grassa mitochondrial DNA.

Neurospora crassa mitochondrial DNA (mtDNA) contains duplications of the tRNA(MMet) gene upstream of a gene (ND2) encoding a subunit of the NADH dehydrogenase complex and of the tRNA(Cys) gene which is found downstream of the apocytochrome b gene. Both duplicated genes are located upstream of the small rRNA gene. The duplications are extended to flanking sequences. In the case of the tRNA(MMet) duplication, two fragments of the ND2 gene are also duplicated. These two fragments, which are not contiguous in the ND2 gene, are connected to each other by a palindromic sequence of 37 bp and together they constitute an open reading frame. The possible involvement of this palindromic sequence in the processes of gene duplication and transfer is discussed. Two overlapping reading frames are present between the tRNA(MMet) and tRNA(Cys) copies. All information of the ND2 duplication and the two overlapping reading frames are present on a polycistronic transcript.

Amino Acid Sequence↗
PubMed2001

An important 2'-OH group for an RNA-protein interaction.

We have investigated the role of 2'-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNA(Cys) and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2'-OH by 2'-deoxy or 2'-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2'-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2'-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2'-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2'-OH group instead provides a monitor of the steric environment during the RNA-synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2'-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

Acylation↗
PubMed2005

Domain-domain communication for tRNA aminoacylation: the importance of covalent connectivity.

Aminoacyl-tRNA synthetases form complexes with tRNA to catalyze transfer of activated amino acids to the 3' end of tRNA. The tRNA synthetase complexes are roughly divided into the activation and tRNA-binding domains of synthetases, which interact with the acceptor and anticodon ends of tRNAs, respectively. Efficient aminoacylation of tRNA by Escherichia coli cysteinyl-tRNA synthetase (CysRS) requires both domains, although the pathways for the long-range domain-domain communication are not well understood. Previous studies show that dissection of tRNA(Cys) into acceptor and anticodon helices seriously reduces the efficiency of aminoacylation, suggesting that communication requires covalent continuity of the tRNA backbone. Here we tested if communication requires the continuity of the synthetase backbone. Two N-terminal fragments and one C-terminal fragment of E. coli CysRS were generated. While the N-terminal fragments were active in adenylate synthesis, they were severely defective in the catalytic efficiency and specificity of tRNA aminoacylation. Conversely, although the C-terminal fragment was not catalytically active, it was able to bind and discriminate tRNA. However, addition of the C-terminal fragment to an N-terminal fragment in trans did not improve the aminoacylation efficiency of the N-terminal fragment to the level of the full-length enzyme. These results emphasize the importance of covalent continuity of both CysRS and tRNA(Cys) for efficient tRNA aminoacylation, and highlight the energetic costs of constraining the tRNA synthetase complex for domain-domain communication. Importantly, this study also provides new insights into the existence of several natural "split" synthetases that are now identified from genomic sequencing projects.

Adenosine Monophosphate↗
PubMed2005

RNA-dependent cysteine biosynthesis in archaea.

Several methanogenic archaea lack cysteinyl-transfer RNA (tRNA) synthetase (CysRS), the essential enzyme that provides Cys-tRNA(Cys) for translation in most organisms. Partial purification of the corresponding activity from Methanocaldococcus jannaschii indicated that tRNA(Cys) becomes acylated with O-phosphoserine (Sep) but not with cysteine. Further analyses identified a class II-type O-phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS). SepRS specifically forms Sep-tRNA(Cys), which is then converted to Cys-tRNA(Cys) by SepCysS. Comparative genomic analyses suggest that this pathway, encoded in all organisms lacking CysRS, can also act as the sole route for cysteine biosynthesis. This was proven for Methanococcus maripaludis, where deletion of the SepRS-encoding gene resulted in cysteine auxotrophy. As the conversions of Sep-tRNA to Cys-tRNA or to selenocysteinyl-tRNA are chemically analogous, the catalytic activity of SepCysS provides a means by which both cysteine and selenocysteine may have originally been added to the genetic code.

Adenosine Triphosphate↗
PubMed1995

Enzymatic aminoacylation of tRNA acceptor stem helices with cysteine is dependent on a single nucleotide.

The discriminator base U73 at the acceptor terminus of Escherichia coli tRNA(Cys) is a determinant for the specific aminoacylation of this tRNA by the cognate cysteine tRNA synthetase. Substitution of U73 has a major deleterious effect on the catalytic efficiency of aminoacylation. Here, we show that an RNA hairpin minihelix and an RNA hairpin microhelix that recreate, respectively, the 12-base pair acceptor-T psi C stem and the 7-base pair acceptor helix of E. coli tRNA(Cys) were aminoacylated with cysteine. As in tRNA(Cys), alteration of U73 to A73, C73, or G73 in the cysteine mini- and microhelices eliminated aminoacylation. This established that the strong influence of U73 on aminoacylation is fully retained from the full-length tRNA(Cys) to the mini- and microhelixCys. Transfer of U73 to the noncognate minihelixAla conferred cysteine acceptance to the latter, despite the presence of the major determinant for alanine tRNA synthetase. Even minihelixGly, which shares U73 with minihelixCys, was an efficient substrate for aminoacylation with cysteine. Conversely, as long as U73 was present in minihelixCys, introduction of the glycine or alanine determinant could not block charging by cysteine tRNA synthetase. Although the catalytic efficiency of aminoacylation of these small RNA helices with cysteine was reduced by orders of magnitude from that of tRNA(Cys), the single nucleotide U73 determines the ability of these RNA helices to be aminoacylated with cysteine. These results demonstrated a dominant role of U73 for aminoacylation of small RNA helices by cysteine tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1993

An unusual RNA tertiary interaction has a role for the specific aminoacylation of a transfer RNA.

The nucleotides in a tRNA that specifically interact with the cognate aminoacyl-tRNA synthetase have been found largely located in the helical stems, the anticodon, or the discriminator base, where they vary from one tRNA to another. The conserved and semiconserved nucleotides that are responsible for the tRNA tertiary structure have been shown to have little role in synthetase recognition. Here we report that aminoacylation of Escherichia coli tRNA(Cys) depends on the anticodon, the discriminator base, and a tertiary interaction between the semiconserved nucleotides at positions 15 and 48. While all other tRNAs contain a purine at position 15 and a complementary pyrimidine at position 48 that establish the tertiary interaction known as the Levitt pair, E. coli tRNA(Cys) has guanosine -15 and -48. Replacement of guanosine -15 or -48 with cytidine virtually eliminates aminoacylation. Structural analyses with chemical probes suggest that guanosine -15 and -48 interact through hydrogen bonds between the exocyclic N-2 and ring N-3 to stabilize the joining of the two long helical stems of the tRNA. This tertiary interaction is different from the traditional base pairing scheme in the Levitt pair, where hydrogen bonds would form between N-1 and O-6. Our results provide evidence for a role of RNA tertiary structure in synthetase recognition.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Synthesis of cysteinyl-tRNACys by a prolyl-tRNA synthetase.

The question of how cysteinyl-tRNA(Cys) is synthesized in organisms that lack a canonical cysteinyl-tRNA synthetase (CysRS) is an important open question in understanding protein synthesis. The prolyl-tRNA synthetase (ProRS) of wide ranging organisms has the ability to mis-activate cysteine without editing. This raises the question of whether the mis-activated cysteine can be charged to tRNA(Cys) to synthesize the correctly matched cys-tRNA(Cys), which may serve as an option in organisms that lack the conventional CysRS. Despite intense searches, such an activity has not been found. Here we show that the ProRS of the radiation-resistant bacterium Deinococcus radiodurans has the ability to charge cysteine to tRNA(Cys) and to retain the cognate pair cys-tRNA(Cys) stably without editing. The cysteinylation by D. radiodurans ProRS is not the major route for synthesis of cys-tRNA(Cys), however, because the organism encodes the conventional CysRS. Nonetheless, the synthesis of cys-tRNA(Cys) in vitro is important and it is unlike previously documented mis-charging of Methanocaldococcus jannaschii ProRS, which synthesizes cys-tRNA(Pro) but not cys-tRNA(Cys). We suggest that the cysteinylation activity of D. radiodruans ProRS may, in the presence of additional factors, offer one option to organisms that lack the conventional CysRS to synthesize cysteinyl-tRNA(Cys) in vivo.

Amino Acids↗
PubMed1996

Nucleotide sequences of nuclear tRNA(Cys) genes from Nicotiana and Arabidopsis and expression in HeLa cell extract.

We have recently characterized Nicotiana cytoplasmic (cyt) tRNA(GCA)Cys as a novel UGA suppressor tRNA. Here we have isolated its corresponding (NtC1) and a variant (NtC2) gene from a genomic library of Nicotiana rustica. Both tRNA(Cys) genes are efficiently transcribed in HeLa cell nuclear extract and yield mature cyt tRNAs(Cys). Sequence analysis of the upstream region of the RAD51 single-copy gene of the Arabidopsis thaliana genome revealed a cluster of three tRNA(Cys) genes which have the same polarity and comprise highly similar flanking sequences. Of the three Arabidopsis tRNA(Cys) genes only one (i.e. AtC2) appears to code for a functional gene which exhibits an almost identical nucleotide sequence to NtC1. These are the first sequenced nuclear tDNAs(Cys) of plant origin.

Arabidopsis↗
PubMed2002

Site-specific biosynthetic incorporation of a fluorescent tag into proteins via cysteine-tRNA(Cys).

Site-specific incorporation of biophysical probes into proteins during translation can permit structure/function studies on selected proteins in heterogeneous environments. We report here a procedure for incorporating a fluorescent tag into proteins via Escherichia coli Cys-tRNA(Cys) during in vitro protein synthesis. Naturally occurring Cys-tRNA(Cys) is an attractive vehicle for fluorophore incorporation since it can be readily prepared in quantity and reacted with commercially available fluorophores. Moreover, proteins can often be constructed with a single Cys so that fluorophore incorporation results in a tag at a unique site.

Base Sequence↗