Biosynthesis of wyosine derivatives in tRNA(Phe) of Archaea: role of a remarkable bifunctional tRNA(Phe):m1G/imG2 methyltransferase.

The presence of tricyclic wyosine derivatives 3'-adjacent to anticodon is a hallmark of tRNA(Phe) in eukaryotes and archaea. In yeast, formation of wybutosine (yW) results from five enzymes acting in a strict sequential order. In archaea, the intermediate compound imG-14 (4-demethylwyosine) is a target of three different enzymes, leading to the formation of distinct wyosine derivatives (yW-86, imG, and imG2). We focus here on a peculiar methyltransferase (aTrm5a) that catalyzes two distinct reactions: N(1)-methylation of guanosine and C(7)-methylation of imG-14, whose function is to allow the production of isowyosine (imG2), an intermediate of the 7-methylwyosine (mimG) biosynthetic pathway. Based on the formation of mesomeric forms of imG-14, a rationale for such dual enzymatic activities is proposed. This bifunctional tRNA:m(1)G/imG2 methyltransferase, acting on two chemically distinct guanosine derivatives located at the same position of tRNA(Phe), is unique to certain archaea and has no homologs in eukaryotes. This enzyme here referred to as Taw22, probably played an important role in the emergence of the multistep biosynthetic pathway of wyosine derivatives in archaea and eukaryotes.

Construction of modified ribosomes for incorporation of D-amino acids into proteins.

While numerous biologically active peptides contain D-amino acids, the elaboration of such species is not carried out by ribosomal synthesis. In fact, the bacterial ribosome discriminates strongly against the incorporation of D-amino acids from D-aminoacyl-tRNAs. To permit the incorporation of D-amino acids into proteins using in vitro protein-synthesizing systems, a strategy has been developed to prepare modified ribosomes containing alterations within the peptidyltransferase center and helix 89 of 23S rRNA. S-30 preparations derived from colonies shown to contain ribosomes with altered 23S rRNAs were found to exhibit enhanced tolerance for D-amino acids and to permit the elaboration of proteins containing D-amino acids at predetermined sites. Five specific amino acids in Escherichia coli dihydrofolate reductase and Photinus pyralis luciferase were replaced with D-phenylalanine and D-methionine, and the specific activities of the resulting enzymes were determined.

Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template.

Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3' end of tRNAs by means of the addition of the essential nucleotides CCA. However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. Here we describe the crystal structure of the Archaeoglobus fulgidus tRNA nucleotidyltransferase in complex with tRNA. We also present ternary complexes of this enzyme with both RNA duplex mimics of the tRNA acceptor stem that terminate with the nucleotides C74 or C75, as well as the appropriate incoming nucleoside 5'-triphosphates. A single nucleotide-binding pocket exists whose specificity for both CTP and ATP is determined by the protein side chain of Arg 224 and backbone phosphates of the tRNA, which are non-complementary to and thus exclude UTP and GTP. Discrimination between CTP or ATP at a given addition step and at termination arises from changes in the size and shape of the nucleotide binding site that is progressively altered by the elongating 3' end of the tRNA.

Yeast tRNA(Phe) expressed in human cells can be selected by HIV-1 for use as a reverse transcription primer.

All naturally occurring human immune deficiency viruses (HIV-1) select and use tRNA(Lys,3) as the primer for reverse transcription. Studies to elucidate the mechanism of tRNA selection from the intracellular milieu have been hampered due to the difficulties in manipulating the endogenous levels of tRNA(Lys,3). We have previously described a mutant HIV-1 with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe) that relies on transfection of yeast tRNA(Phe) for infectivity. To more accurately recapitulate the selection process, a cDNA was designed for the intracellular expression of the yeast tRNA(Phe). Increasing amounts of the plasmid encoding tRNA(Phe) resulted in a corresponding increase in levels of yeast tRNA(Phe) in the cell. The yeast tRNA(Phe) isolated from cells transfected with the cDNA for yeast tRNA(Phe), or in the cell lines expressing yeast tRNA(Phe), were aminoacylated, indicating that the expressed yeast tRNA(Phe) was incorporated into tRNA biogenesis pathways and translation. Increasing the cytoplasmic levels of tRNA(Phe) resulted in increased encapsidation of tRNA(Phe) in viruses with a PBS complementary to tRNA(Phe) (psHIV-Phe) or tRNA(Lys,3) (wild-type HIV-1). Production of infectious psHIV-Phe was dependent on the amount of cotransfected tRNA(Phe) cDNA. Increasing amounts of plasmids encoding yeast tRNA(Phe) produced an increase of infectious psHIV-Phe that plateaued at a level lower than that from the transfection of the wild-type genome, which uses tRNA(Lys,3) as the primer for reverse transcription. Cell lines were generated that expressed yeast tRNA(Phe) at levels approximately 0.1% of that for tRNA(Lys,3). Even with this reduced level of yeast tRNA(Phe), the cell lines complemented psHIV-Phe over background levels. The results of these studies demonstrate that intracellular levels of primer tRNA can have a direct effect on HIV-1 infectivity and further support the role for PBS-tRNA complementarity in the primer selection process.

Selection of retroviral reverse transcription primer is coordinated with tRNA biogenesis.

Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu. To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNA(Phe) to be supplied in trans for infectivity. Wild-type yeast tRNA(Phe) expressed in mammalian cells was transported to the cytoplasm and aminoacylated. In contrast, tRNA(Phe) without the D loop (tRNA(Phe)D(-)) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNA(Phe)D(-) was directly transfected into the cytoplasm of cells. A tRNA(Phe) mutant (tRNA(Phe)UUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNA(Phe). Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis.

Efficient synthesis, termination and release of RNA polymerase III transcripts in Xenopus extracts depleted of La protein.

La proteins are conserved, abundant and predominantly nuclear phosphoproteins which bind to the 3'-U termini of newly synthesized RNA polymerase III transcripts. The human La protein has been implicated in the synthesis, termination and release of such transcripts. Here we examine the potential transcriptional properties of La in Xenopus laevis, using a homologous tRNA gene as template. Immunodepletion of La from cell-free extracts leads to the formation of tRNA precursors lacking 3'-U residues. This shortening can be uncoupled from RNA polymerase III transcription, indicating that it results from nuclease degradation rather than incomplete synthesis. Extracts containing <1% of the normal La protein content synthesize tRNA precursors just as well as complete extracts, with no change in termination efficiency, and the vast majority of these full-length transcripts are not associated with the template or with residual La protein. Hence, Xenopus La seems not to function as an initiation, termination or release factor for RNA polymerase III. Consistent with the recently discovered role of La in yeast tRNA maturation in vivo, recombinant Xenopus La prevents 3'-exonucleolytic degradation of tRNA precursors in vitro. A conserved RNA chaperone function may best explain the abundance of La in eukaryotic nuclei.

A single editing event is a prerequisite for efficient processing of potato mitochondrial phenylalanine tRNA.

In bean, potato, and Oenothera plants, the C encoded at position 4 (C4) in the mitochondrial tRNA Phe GAA gene is converted into a U in the mature tRNA. This nucleotide change corrects a mismatched C4-A69 base pair which appears when the gene sequence is folded into the cloverleaf structure. C-to-U conversions constitute the most common editing events occurring in plant mitochondrial mRNAs. While most of these conversions introduce changes in the amino acids specified by the mRNA and appear to be essential for the synthesis of functional proteins in plant mitochondria, the putative role of mitochondrial tRNA editing has not yet been defined. Since the edited form of the tRNA has the correct secondary and tertiary structures compared with the nonedited form, the two main processes which might be affected by a nucleotide conversion are aminoacylation and maturation. To test these possibilities, we determined the aminoacylation properties of unedited and edited potato mitochondrial tRNAPhe in vitro transcripts, as well as the processing efficiency of in vitro-synthesized potato mitochondrial tRNAPhe precursors. Reverse transcription-PCR amplification of natural precursors followed by cDNA sequencing was also used to investigate the influence of editing on processing. Our results show that C-to-U conversion at position 4 in the potato mitochondrial tRNA Phe GAA is not required for aminoacylation with phenylalanine but is likely to he essential for efficient processing of this tRNA.

Identity of prokaryotic and eukaryotic tRNA(Asp) for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus.

The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements. It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS. This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems. Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs. It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions. Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T. thermophilus AspRS. In the homologous systems, the mutational effects are less pronounced. However, effects in E. coli and T. thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes. Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex.

Rabbit translation elongation factor 1 alpha stimulates the activity of homologous aminoacyl-tRNA synthetase.

Functional and structural sequestration of aminoacyl-tRNA has been recently found in eukaryotic cells and the aminoacyl-tRNA channeling has been suggested [B.S. Negrutskii et al., Proc. Natl. Acad. Sci. 91 (1994) 964-968], but molecular details and mechanism of the process remained unclear. In this paper we have verified a possible interaction between rabbit aminoacyl-tRNA synthetase and homologous translation elongation factor 1 alpha (EF-1 alpha), the proteins which may play a role of sequential components involved in the transfer of the aminoacyl-tRNA along the protein synthetic metabolic chain. The stimulation of the phenylalanyl-tRNA synthetase activity by EF-1 alpha is found. The effect is shown to be specific towards the origin of tRNA and elongation factor molecules. The data obtained favor the direct transfer mechanism of the aminoacyl-tRNA channeling process during eukaryotic protein synthesis.

In vivo deuteration of transfer RNAs: overexpression and large-scale purification of deuterated specific tRNAs.

Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.

Footprinting of tRNA(Phe) transcripts from Thermus thermophilus HB8 with the homologous phenylalanyl-tRNA synthetase reveals a novel mode of interaction.

The phosphates of the tRNA(Phe) transcript from Thermus thermophilus interacting with the cognate synthetase were determined by footprinting. Backbone bond protection against cleavage by iodine of the phosphorothioate-containing transcripts was found in the anticodon stem-loop, the D stem-loop and the acceptor stem and weak protection was also seen in the variable loop. Most of the protected phosphates correspond to regions around known identity elements of tRNA(Phe). Enhancement of cleavage at certain positions indicates bending of tRNAPhe upon binding to the enzyme. When applied to the three-dimensional model of tRNA(Phe) from yeast the majority of the protections occur on the D loop side of the molecule, revealing that phenylalanyl-tRNA synthetase has a rather complex and novel pattern of interaction with tRNAPhe, differing from that of other known class II aminoacyl-tRNA synthetases.

Self-coded 3'-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase.

More than 70% of the RNA synthesized by T7 RNA polymerase during run-off transcription in vitro can be incorrect products, up to twice as long as the expected transcripts. Transcriptions with model templates indicate that false transcription is mainly observed when the correct product cannot form stable secondary structures at the 3'-end. Therefore, the following hypothesis is tested: after leaving the DNA template, the polymerase can bind a transcript to the template site and the 3'-end of the transcript to the product site and extend it, if the 3'-end is not part of a stable secondary structure. Indeed, incubation of purified transcripts with the polymerase in transcription conditions triggers a 3'-end prolongation of the RNA. When two RNAs of different lengths are added to the transcription mix, both generate distinct and specific patterns of prolonged RNA products without any interference, demonstrating the self-coding nature of the prolongation process. Furthermore, sequencing of the high molecular weight transcripts demonstrates that their 5'-ends are precisely defined in sequence, whereas the 3'-ends contain size-variable extensions which show complementarity to the correct transcript. Surprisingly, a reduction of the UTP concentration to 0.2-1.0 mM in the presence of 3.5-4.0 mM of the other NTPs leads to faithful transcription and good yields, irrespective of the nucleotide composition of the template.

Effects of unusual polyamines on phenylalanyl-tRNA formation.

The effects of novel polyamines on aminoacyl-tRNA formation catalyzed by Escherichia coli., Sulfolobus acidocaldarius, and Thermus thermophilus HB8 S-100 extracts were investigated. These effects were diverse and differed depending on the amino acid and the enzyme used. A quaternary polyamine, tetrakis(3-aminopropyl)ammonium, inhibited phenylalanyl-tRNA synthesis catalyzed by the T. thermophilus extract, but not inhibit the other aminoacyl-tRNA formations tested. The inhibition was observed in hybrid reactions where the thermophile tRNA or extract was replaced by its E. coli counterpart, although the quaternary amine did not inhibit Phe-tRNA formation by the E. coli homologous system. Spermine relieved the inhibition of the reaction of thermophile enzyme and tRNA, but not the inhibition of the hybrid reactions. These results suggest that the branched polyamine interacts with both the thermophile enzyme and tRNA(Phe).

Polyamines of sulfur-dependent archaebacteria and their role in protein synthesis.

Several strains of the sulfur-dependent archaebacterium, Sulfolobus, were analyzed for their polyamine content. Caldine (norspermidine), spermidine, and thermine were found to be major components in all of the cells tested. The most abundant polyamine in all cultures examined was spermidine. The Langworthy strain had the highest spermine content, whereas S. acidocaldarius strain no. 7 was devoid of this polyamine. Cultures of strain no. 7 grown at 70 degrees C were rich in spermidine and caldine (triamines) and the thermine: spermidine ratio was much lower than that of cultures grown at 78 degrees C. Equal amounts of thermine and spermidine were present in strain DSM 1616. Preincubation of Langworthy strain extracts at 10 degrees C did not overcome the requirement for polyamines in protein synthesis. Putrescine exerted a concentration-dependent inhibition of the spermine-induced stimulation of protein synthesis at 70 degrees C. Increasing concentrations (6 and 9 mM) of spermine and thermine progressively inhibited poly(U)-dependent phenylalanine incorporation at 45 degrees C to about the same extent, whereas the same concentrations of these polyamines had little effect on the reaction at 70 degrees C. Although 3 mM spermine had only a slight stimulatory effect on the attachment of phenylalanine to tRNA at 65 degrees C, this polyamine had a pronounced effect on the formation of 70S ribosomes in a standard buffer containing 10 mM Mg2+. Increasing the Mg2+ concentration to 30 mM in the absence of spermine was even more effective in causing the reassociation of subunits to form 70S particles.

Influence of ochratoxin B on the ochratoxin A inhibition of phenylalanyl-tRNA formation in vitro and protein synthesis in hepatoma tissue culture cells.

Ochratoxin B (OTB), the dechloro-analogue of ochratoxin A (OTA), was studied separately and in combination with OTA on the aminoacylation of phenylalanine tRNA (tRNAPhe) catalysed by mice liver phenylalanyl-tRNA synthetase. OTB was neither a significant inhibitor of the reaction nor an antagonist of OTA. OTB was also assayed for its possible antagonistic effect on the in vivo protein synthesis inhibition caused by OTA in hepatoma tissue culture cells. No prevention of OTA inhibition could be found for OTB. It rather showed a slight additional inhibitory activity when mixed (100-180 microM) with low concentrations of OTA (40-60 microM). In conclusion, these results are not in favor of an antagonistic effect of OTB with respect to OTA action, at least on the level of cellular protein synthesis.

Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA.

The overall structure of transfer RNA is optimized for its various functions by a series of unique post-transcriptional nucleotide modifications. Since many of these modifications are conserved from prokaryotes through higher eukaryotes, it has been proposed that most modified nucleotides serve to optimize the ability of the tRNA to accurately interact with other components of the protein synthesizing machinery. When a cloned synthetic Escherichia coli tRNAPhe gene was transfected into a bacterial host that carried a defective phenylalanine tRNA-synthetase gene, tRNAPhe was overexpressed by 11-fold. As a result of this overexpression, an undermodified tRNAPhe species was produced that lacked only N6-(delta 2-isopentenyl)-2-methylthioadenosine (ms2i6A), a hypermodified nucleotide found immediately 3' to the anticodon of all major E. coli tRNAs that read UNN codons. To investigate the role of ms2i6A in E. coli tRNA, we compared the aminoacylation kinetics and in vitro codon-reading properties of the ms2i6A-lacking and normal fully modified tRNAPhe species. The results of these experiments indicate that while ms2i6A is not required for normal aminoacylation of tRNAPhe, its presence stabilizes codon-anticodon interaction and thereby prevents misreading of the genetic code.

A synthetic substrate for tRNA splicing.

A novel method for the synthesis of precursor tRNA as substrate for in vitro splicing is reported. A construct consisting of the Saccharomyces cerevisiae pre-tRNAPhe gene under the control of a bacteriophage T7 promoter was assembled from a set of synthetic oligonucleotides and cloned into an M13 vector. By the use of T7 RNA polymerase, BstNI-runoff transcripts were produced. The resulting pre-tRNA was shown to possess mature termini and was accurately spliced by highly purified yeast tRNA-splicing endonuclease and ligase. Using this synthetic pre-tRNA, the kinetic parameters of the tRNA-splicing endonuclease were also determined. Use of this system provides several advantages for the study of tRNA-splicing mechanisms. Mutant tRNA precursors can be readily synthesized. It is also possible to synthesize large quantities of pre-tRNA for structural studies.