tRNA Derivatives Research Articles

The Activities of the Yersinia Protein Kinase A (YpkA) and Outer Protein J (YopJ) Virulence Factors Converge on an eIF2α Kinase

The Yersinia protein kinase A (YpkA) and outer protein J (YopJ) are co-expressed from a single transcript and are injected directly into eukaryotic cells by the plague bacterium Yersinia pestis. When overexpressed in vertebrate or yeast cells, YpkA disrupts the actin-based cytoskeletal system by an unknown mechanism, whereas YopJ obstructs inductive chemokine expression by inhibiting MAPK and NF-kappaB signaling. Previously, we showed that the fission yeast Schizosaccharomyces pombe was sensitive to the kinase activity of YpkA. Here, we screened yeast for cellular processes important for YpkA activity and found that the eIF2alpha kinases mollify the toxicity imparted by the kinase activity of YpkA. Specifically, strains lacking the eIF2alpha kinase Hri2 were particularly sensitive to YpkA. Unexpectedly, the activity of YopJ, which conferred a phenotype consistent with its inhibitory effect on MAPK signaling, was also found to be dependent on Hri2. When expressed in S. pombe, YopJ sensitized cells to osmotic and oxidative stresses through a Hri2-dependent mechanism. However, when co-expressed with YpkA, YopJ protected cells from YpkA-mediated toxicity, and this protection was entirely dependent on Hri2. In contrast, YopJ did not confer protection against the toxic effects of the Yersinia virulence factor YopE. These findings are the first to functionally link YpkA and YopJ and suggest that eIF2alpha kinases, which are critically important in antiviral defenses and protection against environmental stresses, also play a role in bacterial virulence.

The human large subunit ribosomal protein L36A-like contacts the CCA end of P-site bound tRNA

Periodate-oxidized tRNA (tRNAox), the 2′,3′-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3′-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA Aspox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA Asp competed with tRNA Aspox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.

Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry

The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNAIle, the 2',3'-dialdehyde derivative of tRNAIle was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNAIle aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNAIleox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNAIle and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence 601KMSKS605. Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [32P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.

Modeling the reactive properties of tandemly activated tRNAs

Tandemly activated tRNAs, bearing amino acid moieties at both the 2'- and 3'-positions of the 3'-terminal adenosine moiety (A(76)), have been shown to participate efficiently in protein synthesis [B. Wang, J. Zhou, M. Lodder, R. D. Anderson, III and S. M. Hecht, J. Biol. Chem., 2006, 281, 13865]. The mechanism by which such activated tRNAs are able to donate both amino acids to the growing polypeptide chain is not well understood. Here we report the chemical behavior and participation in protein synthesis of new bisaminoacyl derivatives of pdCpA and tRNA. Both amino moieties of the aminoacyl groups are shown to be important to enable participation in protein synthesis; paradoxically, they also confer an unanticipated chemical stability toward different nucleophiles. The results obtained suggest a model for participation of bisaminoacylated tRNAs in protein synthesis.

New photoreactive tRNA derivatives for probing the peptidyl transferase center of the ribosome

Three new photoreactive tRNA derivatives have been synthesized for use as probes of the peptidyl transferase center of the ribosome. In two of these derivatives, the 3' adenosine of yeast tRNA(Phe) has been replaced by either 2-azidodeoxyadenosine or 2-azido-2'-O-methyl adenosine, while in a third the 3'-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNA(Phe) and can be cross-linked to components of the 50S ribosomal subunit by irradiation with near-UV light. Characteristic differences in the cross-linking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the peptidyl transferase center.

Semisynthesis of 3′(2′)‐O‐(Aminoacyl)‐tRNA Derivatives as Ribosomal Substrate

AbstractAn efficient synthesis of (3′‐terminally) 3′(2′)‐O‐aminoacylated pCpA derivatives is described, which could lead to the production of (aminoacyl)‐tRNAs following T4 RNA ligase mediated ligation. The tetrahydrofuranyl (thf) group was used as a permanent protective group for the 2′‐OH of the cytidine moiety which can be removed during the purification of the 3′(2′)‐O‐aminoacylated‐pCpA. This approach allowed for a general synthesis of (3′‐terminally) 3′(2′)‐O‐aminoacylated oligonucleotides. The fully protected pCpA 14 was synthesized by phosphoramidite chemistry and treated with NH3 solution to remove the 2‐cyanoethyl and benzoyl groups (→ 15; Schemes 1 and 2). The 2′‐O‐thf‐protected‐pCpA 15 was coupled with α‐amino acid cyanomethyl esters, and the products 20a–c were deprotected and purified with AcOH buffer to afford 3′(2′)‐O‐aminoacylated pCpA 21a–c in high yields. The 3′(2′)‐O‐aminoacylated pCpA were efficiently ligated with tRNA(− CA) to yield (aminoacyl)‐tRNA which was an active substrate for the ribosome.

Escherichia coli Ribosomes as a Model for Testing New Photoactivated tRNA Analogs Containing 6-Thioguanosine Residues

A comparative study of photoreactive tRNA derivatives containing a 6-thioguanosine or diazirine derivative of 5-methyleneaminouridine was performed using Escherichia coli ribosomes. The conditions of producing functional tRNAs containing photoreactive residues have been selected using T7 RNA polymerase transcription. Photochemical modification was conducted upon binding the corresponding tRNAPhe transcripts carrying the photoreactive residues of nucleotide analogs to the P site of the ribosome. The results of modification were analyzed using oligonucleotide-directed hydrolysis of rRNA by RNase H, as well as reverse transcription. It is shown that 6-thioguanosine is a promising agent for studying RNA–protein complexes by photochemical crosslinking methods.

Nuclear DNA-encoded tRNAs targeted into mitochondria can rescue a mitochondrial DNA mutation associated with the MERRF syndrome in cultured human cells.

Mitochondrial DNA (mtDNA) mutations are an important cause of human disease for which there is no efficient treatment. Our aim was to determine whether the A8344G mitochondrial tRNA(Lys) mutation, which can cause the MERRF (myoclonic epilepsy with ragged-red fibers) syndrome, could be complemented by targeting tRNAs into mitochondria from the cytosol. Import of small RNAs into mitochondria has been demonstrated in many organisms, including protozoans, plants, fungi and animals. Although human mitochondria do not import tRNAs in vivo, we previously demonstrated that some yeast tRNA derivatives can be imported into isolated human mitochondria. We show here that yeast tRNALys derivatives expressed in immortalized human cells and in primary human fibroblasts are partially imported into mitochondria. Imported tRNAs are correctly aminoacylated and are able to participate in mitochondrial translation. In transmitochondrial cybrid cells and in patient-derived fibroblasts bearing the MERRF mutation, import of tRNALys is accompanied by a partial rescue of mitochondrial functions affected by the mutation such as mitochondrial translation, activity of respiratory complexes, electrochemical potential across the mitochondrial membrane and respiration rate. Import of a tRNALys with a mutation in the anticodon preventing recognition of the lysine codons does not lead to any rescue, whereas downregulation of the transgenic tRNAs by small interfering RNA (siRNA) transiently abolishes the functional rescue, showing that this rescue is due to the import. These findings prove for the first time the functionality of imported tRNAs in human mitochondria in vivo and highlight the potential for exploiting the RNA import pathway to treat patients with mtDNA diseases.

Prokaryotic and eukaryotic tetrameric phenylalanyl-tRNA synthetases display conservation of the binding mode of the tRNA(Phe) CCA end.

The interaction of human phenylalanyl-tRNA synthetase, a eukaryotic prototype with an unknown three-dimensional structure, with the tRNA(Phe) acceptor end was studied by s(4)U-induced affinity cross-linking with human tRNA(Phe) derivatives site-specifically substituted at the single-stranded 3' end. Two different subunits of the enzyme bind two adjacent nucleotides of the tRNA(Phe) 3' end: nucleotide 76 is associated with the catalytic alpha subunit, while nucleotide 75 is in contact with the beta subunit. The binding mode is similar to that revealed previously in structural and affinity cross-linking studies of the prokaryotic Thermus thermophilus phenylalanyl-tRNA synthetase. Our results suggest that the distinctive features of tRNA(Phe) acceptor end binding are conserved for the eukaryotic and prokaryotic tetrameric phenylalanyl-tRNA synthetases despite their significant differences in the domain composition of the beta subunits. The data from affinity cross-linking experiments with human phenylalanyl-tRNA synthetase complexed with small ligands (ATP and/or phenylalanine or a stable synthetic analogue of phenylalanyl adenylate) reveal that the location of the tRNA(Phe) acceptor end varies with the presence and nature of other substrates. The lack of substrate activity of human tRNA(Phe) substituted with s(4)U at the 3'-terminal position suggests that base-specific interactions of the terminal adenosine are critically important for a productive interaction. The conformational rearrangement of the tRNA 3' end induced by the other substrates and dictated by base-specific contacts of the terminal nucleotide is an additional means of ensuring the phenylalanylation specificity in both prokaryotic and eukaryotic systems.

TRNA 1Ser(G 34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons

The tRNA 1 Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli. Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon. The tRNA 1 Ser(G 34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons. This means that the formation of G–A or G–G pair allowed at the wobble position and these base pairs are noncanonical. The translational efficiency of the tRNA 1 Ser(G 34) for UCA or UCG codon depends on the 2′- O-methylation of the C 32 (Cm). The 2′- O-methylation of C 32 may give rise to the space necessary for G–A or G–G base pair formation between the first position of anticodon and the third position of codon.

Interactions between 23S rRNA and tRNA in the ribosomal E site.

Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.

Transit of tRNA through the Escherichia coliRibosome: CROSS-LINKING OF THE 3′ END OF tRNA TO SPECIFIC NUCLEOTIDES OF THE 23 S RIBOSOMAL RNA AT THE A, P, AND E SITES

When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at the 3' terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3' termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3'-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA(Phe), remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3'-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.

Suppression of mutations in mitochondrial DNA by tRNAs imported from the cytoplasm.

Mitochondrial import of a cytoplasmic transfer RNA (tRNA) in yeast requires the preprotein import machinery and cytosolic factors. We investigated whether the tRNA import pathway can be used to correct respiratory deficiencies due to mutations in the mitochondrial DNA and whether this system can be transferred into human cells. We show that cytoplasmic tRNAs with altered aminoacylation identity can be specifically targeted to the mitochondria and participate in mitochondrial translation. We also show that human mitochondria, which do not normally import tRNAs, are able to internalize yeast tRNA derivatives in vitro and that this import requires an essential yeast import factor.

Mapping Contacts between Escherichia coli Alanyl tRNA Synthetase and 2‘ Hydroxyls Using a Complete tRNA Molecule

A dual-specific derivative of yeast tRNA(Phe) is described whose features facilitate structure-function studies of tRNAs. This tRNA has been made in three different bimolecular forms that allow modifications to be easily introduced into any position within the molecule. A set of deoxynucleotide substituted versions of this tRNA has been created and used to examine contacts between tRNA and Escherichia coli alanyl-tRNA synthetase, an enzyme previously shown to interact with 2'-hydroxyls in the acceptor stem of the tRNA. Because the present experiments used a full-length tRNA, several contacts were identified that had not been previously found using microhelix substrates. Contacts at similar sites in the T-loop are seen in the cocrystal structure of tRNA(Ser) and Thermus thermophilus seryl-tRNA synthetase.

Expression of bovine mitochondrial tRNASer GCU derivatives in Escherichia coli.

By replacing a stretch of five A-U base pairs in the acceptor stem with G-C pairs, mitochondrial tRNA-SerGCU lacking a D arm could be expressed in Escherichia coli cells in considerable amounts. The expressed tRNA with no modified nucleoside was serylated in vitro with the mitochondrial enzyme. The tRNASerGCU derivatives carrying identity elements for alanine tRNA and the related anticodons were expressed. However, this expression event did not affect cell growth, probably because the expression started from the late log phase, which suggests that these mitochondrial tRNA derivatives are not involved in E.coli gene expression systems. Although there are some restrictions in the secondary structure of tRNAs that can be expressed by this method, it could prove useful for preparing large amounts of heterologous tRNAs in vivo.

Interaction of human immunodeficiency virus type 1 reverse transcriptase with primer tRNALys3 and affinity modification of the enzyme by tRNALys3 derivatives.

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity. In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Kd values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.

Mechanism of Action of Streptogramins and Macrolides

Protein synthesis is catalysed by ribosomes and cytoplasmic factors. Bacterial ribosomes (70S) are made up of 2 subunits (50S and 30S) containing ribosomal RNA (rRNA) and ribosomal proteins: the 30S binds messenger RNA and begins the ribosomal cycle (initiation), whereas 50S binds transfer RNA (tRNA) derivatives and controls elongation. The key reaction, peptide bond formation, is promoted by the catalytic centre of 50S (the peptidyl transferase centre), and the growing peptide chain (peptidyl-tRNA) attached at the donor P site undergoes peptide linkage with an aminoacyl-tRNA at the acceptor A site. This reaction is inhibited by several antibiotics, the best known being chloramphenicol, and the macrolide-lincosamide-streptogramin (MLS) group. These inhibitors have a reversible action, except for streptogramins that are composed of A and B components, which are bacteriostatic alone, but bactericidal when combined. The peptidyl transferase centre has been identified at the 50S surface, and the binding sites of inhibitors have been mapped within this domain: some of these sites overlap (e.g. those of macrolides, and type B streptogramins, which compete for binding to ribosomes). Chloramphenicol blocks the catalytic portion, and A streptogramins the substrate sites of the peptidyl transferase centre. Macrolides and type B streptogramins interfere with the formation of long polypeptides and cause a premature detachment of incomplete peptide chains. The synergism between types A and B streptogramins is due to induction by type A streptogramins of an increased ribosome affinity for type B streptogramins. Microbial resistance to antibiotics mainly involves inactivation of inhibitors and modification of targets (mutations of ribosomal proteins or rRNA genes). Alterations of rRNA bases can induce resistance to a single inhibitor or to a group of antibiotics (e.g. MLSB). The impact of resistance in chemotherapy is less important for streptogramins than for other inhibitors, because the synergistic effect of A and B streptogramins also applies to strains resistant to the MLSB group. It is proposed that mutations and modifications of rRNA bases induce conformational ribosomal changes that prevent antibiotics binding to the target. Conformational changes are also triggered by type A streptogramins: they are responsible for their synergism with type B streptogramins.

Initial Binding of the Elongation Factor Tu·GTP·Aminoacyl-tRNA Complex Preceding Codon Recognition on the Ribosome

The first step in the sequence of interactions between the ribosome and the complex of elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA, which eventually leads to A site-bound aminoacyl-tRNA, is the codon-independent formation of an initial complex. We have characterized the initial binding and the resulting complex by time-resolved (stopped-flow) and steady-state fluorescence measurements using several fluorescent tRNA derivatives. The complex is labile, with rate constants of 6 x 10(7) M-1 s-1 and 24 s-1 (20 degrees C, 10 mM Mg2+) for binding and dissociation, respectively. Both thermodynamic and activation parameters of initial binding were determined, and five Mg2+ ions were estimated to participate in the interaction. While a cognate ternary complex proceeds form initial binding through codon recognition to rapid GTP hydrolysis, the rate constant of GTP hydrolysis in the non-cognate complex is 4 orders of magnitude lower, despite the rapid formation of the initial complex in both cases. Hence, the ribosome-induced GTP hydrolysis by EF-Tu is strongly affected by the presence of the tRNA. This suggests that codon-anticodon recognition, which takes place after the formation of the initial binding complex, provides a specific signal that triggers fast GTP hydrolysis by EF-Tu on the ribosome.

Repression of Vertebrate RNA Polymerase III Transcription by DNA Binding Proteins Located Upstream from the Transcription Start Site

Derivatives of yeast tRNA and XenopustRNA and 5 S RNA genes have been constructed in which natural 5′ flanking sequences have been replaced by the binding sites for either the yeast transcription activator protein GCN4 or the three amino-terminal zinc fingers of the Xenopusfactor TFIIIA (zf1–3). The binding sites for these proteins have been placed at various distances upstream from the start site for transcription initiation in the parent genes. Each of these plasmid DNAs is actively transcribed in both an unfractionated transcription extract prepared from unfertilized Xenopuseggs and in a reconstituted Xenopustranscription system. Binding of the test proteins to plasmid DNAs harbouring the cognate binding sites severely represses transcription when these binding sites are located less than approximately 40 base-pairs upstream from the transcription start site. The DNA-binding proteins are without effect on the transcription of plasmids lacking binding sites or when the binding sites are located further upstream. Assembly of DNA templates into a complete transcription complex prior to addition of the DNA-binding proteins prevents repression. Proteins present in a fraction containing TFIIIB are necessary for this reversal of repression. These data suggest that vertebrate TFIIIB binds upstream from class III genes and this binding can be prevented by occlusion of the TFIIIB binding site by the test proteins GCN4 and zf1–3. f2 f2 Abbreviations used: polIII, polymerase III; TBP, TATA box binding protein; TAF, TBP-associated factor.

Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs

Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells.