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PubMed2004

Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1.

The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA(Gln), that AspRS is nondiscriminating, and that its Asp-tRNA(Asn) product is transamidated by AdT. On the other hand, tRNA(Gln) is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.

Amino Acid Sequence↗
PubMed1992

Synthetic human tRNA(UUULys3) and natural bovine tRNA(UUULys3) interact with HIV-1 reverse transcriptase and serve as specific primers for retroviral cDNA synthesis.

Full-length and 5'-truncated variants of human (h) tRNA(UUULys3) were synthesized by in vitro transcription using SP6 RNA polymerase. Bovine(b) tRNA(SUULys3) was purified from calf liver. Both full-length tRNA species were shown to be biologically active in an aminoacylation assay. Gel retardation assays revealed that both full-length tRNA species, as well as a 5'-truncated h-tRNA(UUULys3) molecule containing 24 nucleotides (nt) at the 3' end (Lys24), interact with human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Competition studies with these three tRNA species demonstrate that the 3' end of h-tRNA(UUULys3) contributes to the interaction with HIV-1 RT. Escherichia coli tRNA(UUULys) and tRNA(UUCGlu2) were also able to interact with the enzyme, whereas unrelated RNA molecules such as E. coli 5S rRNA did not bind to RT. Both b-tRNA(SUULys3) and h-tRNA(UUULys3) molecules, as well as the 5'-truncated variants, could be demonstrated to prime cDNA synthesis specifically using a HIV-1 RNA template, prepared by in vitro transcription, indicating that other viral or cellular proteins are not essential for this process. E. coli tRNA(UUULys) and tRNA(UUCGlu2), although able to interact with HIV-1 RT, failed to prime retroviral transcription. Products of cDNA synthesis were characterized by polymerase chain reaction, demonstrating that at least 18 nt at the 3' ends of h-tRNA(UUULys3) and b-tRNA(SUULys3) are still present in the cDNA product, whereas the 5' ends of both primer molecules were removed by the RNase H activity of HIV-1 RT.

Animals↗
PubMed1996

Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet).

Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.

Archaea↗
PubMed1989

A human tRNA gene cluster encoding the major and minor valine tRNAs and a lysine tRNA.

A human genomic DNA clone hybridizing to mammalian valine tRNA(IAC) contained a cluster of three tRNA genes. Two valine tRNA genes with anticodons of AAC and CAC, encoding the major and minor cytoplasmic valine tRNA isoacceptors, respectively, and a lysine tRNA(CUU) gene were identified by Southern blot hybridization and DNA sequence analysis of a 7.1-kb region. At least nine Alu family members were interspersed throughout the 18.5-kb human DNA fragment, with three Alu elements in the intergenic region between the valine tRNA(AAC) gene and the lysine tRNA gene. Each of the five Alu family members in the sequenced region can be categorized into one of the four Alu subfamilies. The coding regions of all three tRNA genes contain characteristic internal split promoter sequences and typical RNA polymerase III termination signals in the 3'-flanking regions. The tRNA genes are accurately transcribed by RNA polymerase III in a HeLa cell extract, since the RNase T1 fingerprints of the mature-sized tRNA transcription products are consistent with the structural genes. The lysine tRNA(CUU) gene was transcribed only slightly more efficiently than the valine tRNA(CAC) gene in the homologous in vitro transcription system. Surprisingly, the valine tRNA(CAC) gene was transcribed about eightfold more efficiently than the valine tRNA(AAC) gene, implicating the presence of a modulatory element in the upstream region flanking the tRNA(CAC) gene.

Base Sequence↗
PubMed1994

Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes.

Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10. When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C. When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C. The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay. At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type. The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser). Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity. Modified nucleosides of these tRNAs were also analyzed.

Anticodon↗
PubMed1992

Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain.

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.

Arabidopsis↗
PubMed2001

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.

Animals↗
PubMed1979

Separation of functionally distinct regions of a macromolecular substrate. Stimulation of tRNA nucleotidyltransferase by a nonreacting fragment of tRNA.

Rabbit liver tRNA nucleotidyltransferase catalyzes the incorporation of AMP and CMP into the model acceptor substrate, cytidine. The apparent Km for cytidine in this reaction is about 80 to 90 mM which is more than 10(4) greater than the Km values for the natural substrates, tRNA lacking the terminal AMP (tRNA-C-C) and tRNA lacking the terminal pCpA (tRNA-C). The Vmax values for the model reaction are only 5% and 2% of those for the reaction with the natural tRNA substrates. Addition of the tRNA fragments, tRNA lacking the terminal XpCpCpA sequence (tRNA-(X - 1)p) and tRNA lacking the terminal CpCpA (tRNA-Xp), greatly stimulates the rate of nucleotide incorporation into cytidine. In the case of CMP incorporation into cytidine, tRNA-Xp stimulates the reaction about 60-fold, to a rate similar to that of the normal reaction with tRNA-C. The tRNA fragment has no effect on the apparent Km of either cytidine or CTP, but only alters the Vmax of the reaction. Stimulation of the model reactions is maximal with tRNA fragments of specific chain lengths. These results provide direct evidence that the nonreacting regions of a substrate molecule play an important role in the catalytic efficiency of an enzyme.

Adenosine Monophosphate↗
PubMed2000

The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication.

The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.

Acylation↗
PubMed1993

Reaction of modified and unmodified tRNA(Tyr) substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus).

Three species of tRNA(Tyr) have been examined as substrates for the transfer reaction of the tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus: Escherichia coli tRNA(Tyr), B. stearothermophilus tRNA(Tyr) expressed in E. coli, and B. stearothermophilus tRNA(Tyr) that has been transcribed in vitro. The binding of the first two substrates to TyrRS may be readily monitored by stopped-flow studies of tryptophan fluorescence to give the rate and equilibrium constants. The in vitro-transcribed tRNA(Tyr), which lacks the modified bases queuosine and 2-(methylthio)-N6-isopentenyladenosine in the anticodon loop, does not cause a significant change in tryptophan fluorescence upon binding. The three tRNA(Tyr) substrates exhibit very similar steady-state kinetics in the charging reaction. Pre-steady-state kinetics of the transfer reaction, monitored by stopped-flow measurements of the change in protein fluorescence on the addition of tRNA(Tyr) to the E.Tyr-AMP complex, show two exponential changes for the modified tRNA(Tyr) substrates. The first is that due to substrate binding. The second has an identical rate to the single change observed for the reaction with the in vitro-transcribed tRNA(Tyr) and to that monitored by quenched-flow measurements on the formation of Tyr-tRNA(Tyr). Hence, the transfer reaction can be observed by stopped-flow. The dissociation constants (KtRNA) of tRNA from the enzyme and rates of tyrosine transfer (k4) show that all three tRNA molecules are kinetically equivalent substrates for TyrRS. The value of k4 is also similar to that found for authentic tRNA(Tyr) from B. stearothermophilus.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1976

Status of tRNA charging, trinucleotide acceptor sequence and tRNA nucleotidyltransferase activity in the human placenta.

Samples of tRNA isolated from the cell sap of full-term human placenta were found to have a low capacity for accepting amino acids in the presence of partially purified synthetase preparations made from placental or rat liver cell sap. Gel electrophoresis of placental tRNA showed that part of this could be accounted for by gross degradation. The proportion of chargeable tRNA carrying amino acids was estimated by periodate oxidation followed by stripping and then charging with labeled amino acids. Only 50% of chargeable placental tRNA was in the charged state when isolated, whereas 87% of freshly isolated rat liver tRNA was found to be charged with amino acids. A fraction from placental cell sap was shown to have tRNA nucleotidyltransferase activity. When placental tRNA was incubated with this fraction and [3H]ATP or [3H]CTP, ATP was incorporated into about 12% of the tRNA molecules and CTP into 5-7%. When rat liver tRNA was used in place of placental tRNA, [3H]ATP was incorporated into less than 5% of the tRNA molecules. By using snake-venom diesterase over short periods of incubation, it was confirmed that the ATP had been incorporated terminally as AMP into the placental tRNA. These observations show that, in contrast to rat liver tRNA, tRNA prepared from human placenta is poorly charged with amino acids, many of the molecules lack the acceptor trinucleotide and there is extensive degradation beyond this stage.

Amino Acyl-tRNA Synthetases↗
PubMed2006

Seasonal differences in activities of rabbit liver tRNA and aminoacyl-tRNA synthetases specific for valine and arginine under myocardial ischemia.

UNLABELLED: The objective of this study was to examine the acceptor activities of tRNA for amino acids, valine and arginine, and the activities of the corresponding aminoacyl-tRNA synthetases of normal rabbit liver and 6, 12 and 24 h after experimental myocardial ischemia in different seasons of the year. MATERIAL AND METHODS: Male rabbits (2.5-3.5 kg) were used. Acute myocardial ischemia was induced by occlusion of the left anterior descending coronary artery. tRNA and aminoacyl-tRNA synthetases were isolated from control rabbit liver and 6, 12 and 24 h after experimental myocardial ischemia in autumn (September and October) and winter (December and January). The acceptor activity of tRNA and the activity of valyl- and arginyl-tRNA synthetases were determined using 14C-labeled amino acids, valine and arginine. RESULTS: The results showed that acceptor activity of rabbit liver tRNA for valine and arginine under 6, 12 and 24 h experimental myocardial ischemia in autumn was higher by 24-35% than in winter. Activities of rabbit liver valyl- and arginyl-tRNA synthetases under 6, 12 and 24 h experimental myocardial ischemia in autumn were lower by 15-32% than in winter. No differences in the activity of tRNA and aminoacyl-tRNA synthetases between control groups of both seasons were observed. CONCLUSIONS: The experimental data suggest that acceptor activity of rabbit liver tRNA for valine and arginine and activity of valyl- and arginyl-tRNA synthetases under 6, 12 and 24 h experimental myocardial ischemia are different in autumn and winter. The decrease of acceptor activity of tRNA for valine and arginine after experimental myocardial ischemia correlates with an increase in the activity of valyl- and arginyl-tRNA synthetases both in autumn and winter. It may be part of the compensatory mechanism of the cell to keep synthesis of protein in a normal range under extreme conditions.

Acute Disease↗
PubMed1992

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.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Analysis of murine leukemia virus replication complemented by yeast tRNA(Phe) reveals inherent preferences for the tRNA primer selected for reverse transcription.

The replication of murine leukemia virus (MuLV) requires the capture of a cellular tRNA(Pro) as a primer for reverse transcription. To further study the specificity of primer selection, we have utilized a defective MuLV in which the primer-binding site (PBS) has been altered to be complementary to a nonmammalian tRNA, yeast tRNA(Phe). Infectivity of the defective MuLV is dependent upon co-expression of yeast tRNA(Phe) in the cell. Defective MuLV genomes have been constructed in which the PBS was altered to be complementary to tRNA(Phe) that also encoded the cDNA for tRNA(Phe). Transfection of these defective proviral genomes into cells resulted in the production of infectious MuLV as determined by a single-round assay. The amount of infectious virus produced using this complementation system, though, was approximately 6-fold lower than that produced following transfection of defective proviral genomes with a wild-type PBS complementary to tRNA(Pro). The lower infectivity was not due to reduced expression of tRNA(Phe) in the transfected cells as compared to endogenous tRNA(Pro) or tRNA(Lys,3). Serial passage of the MuLV genome with a PBS complementary to tRNA(Phe) that encoded tRNA(Phe) resulted in amplification of the virus. Using this rescue system, we have passaged the virus for four serial passages, after which time a revertant genome in which the PBS was altered to be complementary to tRNA(Gln) was detected that grew to high titers following subsequent serial passage. The results of these studies suggest that MuLV has preferences for the tRNA primer used in reverse transcription and are discussed with respect to the mechanism of primer selection.

Base Sequence↗
PubMed1990

Nucleotides that determine Escherichia coli tRNA(Arg) and tRNA(Lys) acceptor identities revealed by analyses of mutant opal and amber suppressor tRNAs.

We have constructed an opal suppressor system in Escherichia coli to complement an existing amber suppressor system to study the structural basis of tRNA acceptor identity, particularly the role of middle anticodon nucleotide at position 35. The opal suppressor tRNA contains a UCA anticodon and the mRNA of the suppressed protein (which is easily purified and sequenced) contains a UGA nonsense triplet. Opal suppressor tRNAs of two tRNA(Arg) isoacceptor sequences each gave arginine in the suppressed protein, while the corresponding amber suppressors with U35 in their CUA anticodons each gave arginine plus a second amino acid in the suppressed protein. Since C35 but not U35 is present in the anticodon of wild-type tRNA(Arg) molecules, while the first anticodon position contains either C34 or U34, these results establish that C35 contributes to tRNA(Arg) acceptor identity. Initial characterizations of opal suppressor tRNA(Arg) mutants by suppression efficiency measurements suggest that the fourth nucleotide from the 3' end of tRNA(Arg) (A73 or G73 in different isoacceptors) also contributes to tRNA(Arg) acceptor identity. Wild-type and mutant versions of opal and amber tRNA(Lys) suppressors were examined, revealing that U35 and A73 are important determinants of tRNA(Lys) acceptor identity. Several possibilities are discussed for the general significance of having tRNA acceptor identity in the same positions in different tRNA acceptor types, as exemplified by positions 35 and 73 in tRNA(Arg) and tRNA(Lys).

Anticodon↗
PubMed1992

Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay.

Although the reverse transcriptase (RT) of human immunodeficiency virus (HIV) uses human tRNA(3Lys) as a primer of viral genome DNA synthesis in vivo, HIV RT binds Escherichia coli glutamine tRNA and in vitro-made human lysine tRNA with nearly equivalent affinities. We show that HIV RT can use either tRNA(3Lys) or tRNA(2Gln) as a primer for DNA synthesis in vitro without the addition of any other host or viral proteins. E. coli tRNA(2Gln) can serve as a primer for HIV RT if a primer-binding site sequence complementary to the 3' end of tRNA(2Gln) is at the 3' end of the template. With this reduced template, the specificity of binding the proper tRNA is due to base-pairing between a bound tRNA to the primer-binding site of the viral RNA template rather than sequence-specific recognition of tRNA(3Lys) by RT. If an 8-nucleotide viral sequence 3' to the primer-binding site is included in the template, then addition of Zn2+ or Co2+ is required for tRNA(3Lys)-primed synthesis, and tRNA(2Gln) now fails to prime synthesis. The latter result implies that a template sequence adjacent to the primer-binding site and containing 6 nucleotides complementary to the anticodon loop of human tRNA(3Lys) plays an active role in tRNA discrimination.

Base Sequence↗
PubMed1994

Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

Animals↗
PubMed1994

Utilization of selenocysteyl-tRNA[Ser]Sec and seryl-tRNA[Ser]Sec in protein synthesis.

The UGA selenocysteine (Sec) codon in glutathione peroxidase mRNA and in selenoprotein P and the UGA stop codon in rabbit beta-globin mRNA were employed to study the utilization of Sec-tRNA[Ser]Sec and Ser-tRNA[Ser]Sec in protein synthesis. In vitro Ser-tRNA[Ser]Sec served as a suppressor of the UGA Sec codon as well as the UGA stop codon, while Sec-tRNA[Ser]Sec did not. However, in vivo Sec-tRNA[Ser]Sec did donate Sec to glutathione peroxidase in Xenopus oocytes microinjected with glutathione peroxidase mRNA and Sec-tRNA. A ribosome binding assay was devised to investigate the interaction of aminoacyl-tRNA, rabbit reticulocyte ribosomes, and eukaryotic elongation factor 1 (eEF-1) in response to the appropriate trinucleoside diphosphate template. Ser-tRNA[Ser]Sec bound weakly to ribosomes in the presence of eEF-1 and UGA as compared to Phe-tRNA, Ser-tRNAIGA, and Met-tRNAm which bound more efficiently in the presence of eEF-1 and the appropriate template. No increase in the binding of Sec-tRNA[Ser]Sec was observed under the same conditions as Ser-tRNA[Ser]Sec. The ribosome binding studies substantiated the finding that Ser-tRNA[Ser]Sec serves as a suppressor of UGA codons in protein synthesis, but Sec-tRNA[Ser]Sec does not. In addition, these studies provide strong evidence that a specific elongation factor is required in mammalian cells for insertion of Sec into protein from Sec-tRNA[Ser]Sec.

Acylation↗