Isoleucylation of transfer RNA f Met (E. coli) by isoleucyl-transfer RNA synthetase from Escherichia coli.
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The non-enzymatic specific amino-acylation of transfer RNA at high pressure.
This paper shows that the phenylalanine-specific tRNA of Escherichia coli as well as the yellow lupin methionine initiator tRNAMet can be charged specifically with phenylalanine and methionine, respectively, in the absence of specific aminoacyl-tRNA synthetases, under high pressure of a maximum of 6 kbar (1 bar = 10(5) Pa; 1 atm = 1.01 x 10(5) Pa). The esterification reaction takes places at the 3' end of the tRNA molecules. The yield of Phe-tRNAPhe or Met-tRNAMet at high pressure is approximately 10 times lower than that of the enzymatic aminoacylation reaction. This reaction seems to be specific, and mis-aminoacylation of tRNAPhe and tRNAMet with serine is negligible. It is well known that tRNA undergoes conformational changes during interaction with an aminoacyl-tRNA synthetase. Similarly, on the basis of circular dichroism spectra, we showed that the conformation of tRNA at high pressure differs slightly from its original A-RNA form. Therefore, it can be speculated that the chargeable conformation of tRNA induced by the aminoacyl-tRNA synthetase during enzymatic aminoacylation and the one created at high pressure are similar and are most probably formed by a dehydration mechanism. We think that the 'unique' tertiary structure of tRNA existing under high pressure creates an active centre which might itself catalyse ester bond formation. Therefore, the structure of the amino acid stem of tRNA may determine (code) the charging of the particular amino acid to specific tRNA. This code is clearly distinct from the rules of the classical genetic code.
Acylation↗
Nucleotide and aminoacyl-tRNA specificity of the mammalian mitochondrial elongation factor EF-Tu.Ts complex.
The bovine mitochondrial elongation factor Tu.Ts complex (EF-Tu.Tsmt) promotes the binding of aminoacyl-tRNA to ribosomes. In the presence of GTP, this complex functions catalytically. Both dGTP and ddGTP can replace GTP although about 4-fold higher concentrations are required. ATP, CTP and UTP are not active. ITP can replace GTP when used at 10- to 20-fold higher concentrations. The catalytic use of EF-Tu.Tsmt is inhibited by GDP but not by GMP. XDP also inhibits although about 20-fold higher concentrations are required. EF-Tu.Tsmt will promote the binding of Phe-tRNA to either Escherichia coli or mitochondrial ribosomes. Unlike E. coli EF-Tu, EF-Tu.Tsmt will promote the binding of AcPhe-tRNA to ribosomes about 25% as efficiently as Phe-tRNA. EF-Tu.Tsmt is active in catalyzing the binding of E. coli Met-tRNAmmet to ribosomes. EF-Tu.Tsmt has about 30% as much activity with E. coli Met-tRNAimet but has essentially no activity with E. coli fMet-tRNAimet. Neither yeast Met-tRNAimet nor fMet-tRNAimet is recognized by bovine EF-Tu.Tsmt.
Acylation↗
Interaction of yeast transcription factor IIIC with dimeric Schizosaccharomyces pombe tRNA(Ser)-tRNA(Met) genes.
A unique tRNA(Ser)-tRNA(Met) tandem gene arrangement was characterized previously from Schizosaccharomyces pombe. Three alleles exist in which a tRNA(Ser) gene is separated by 7 base pairs from an initiator tRNA(Met) gene. Promotion of transcription occurs only within the tRNA(Ser) gene, yielding a dimeric precursor transcript. Using nuclease protection and gel retardation assays, we have analyzed how the Saccharomyces cerevisiae RNA polymerase III transcription factor C (TFIIIC) interacts with this dimeric gene template. The primary interaction site of TFIIIC with the tRNA(Ser) gene is at the 3'-internal control region (ICR), which can be distinguished kinetically from its weaker interaction with the 5'-ICR of the gene. We examined a variety of point mutations and double mutations within the tRNA(Ser) gene which reduce transcription. We found that changes in highly conserved nucleotides within the ICRs reduce TFIIIC binding up to 7-fold compared with the parent suppressor gene. The interaction of TFIIIC with the tRNA(Ser) gene does not sterically prevent stable binding of TFIIIC to the 3'-ICR of the tRNA(Met) gene. However, the affinity of binding of TFIIIC to the dimeric template is 7-fold higher than to the tRNA(Met) gene, alone, demonstrating that the tRNA(Met) gene contains intrinsically weak promoter elements. This may contribute to the inability of the tRNA(Met) gene to independently direct transcription from its ICR elements.
Alleles↗
Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts: a comparative study.
Retroviral gene delivery systems for RNA polymerase II (RNA pol II)-based promoters have been developed and are widely used in gene transfer studies. In contrast, gene delivery systems with RNA pol III-based expression cassettes have not been studied comprehensively, although therapeutic applications (e.g., ribozymes, antisense, triplex RNA and RNA decoys) have been proposed. In this report, we describe retroviral vectors designed to optimize expression of short chimeric RNAs transcribed from a number of RNA pol III promoters. Our results show that all analysed RNA pol III expression cassettes (tRNA, U6, Ad VA1), regardless of orientation, do not transcribe efficiently when located between the retroviral long terminal repeats (LTRs). In contrast, high steady-state expression levels can be achieved by inserting the RNA pol III expression cassette into the U3 region of the LTR (double-copy design). Compared to human tRNA gene promoters (tRNA(Met), tRNA(Val)), the human small nuclear RNA U6 gene (U6) and the adenovirus virus-associated RNA 1 (Ad VA1) gene promoters yielded higher expression levels. The majority of the chimeric U6-derived transcripts were detected in the nuclear RNA fraction, and the VA1 and tRNA-driven transcripts were predominantly detected in the cytoplasmic compartments. This report is the first comparative study of RNA pol III-driven promoters expressing short chimeric transcripts leading to an optimized retroviral-vector design.
Adenoviridae↗
Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli.
The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA(Ser)(CGA), an Arabidopsis intron-containing tRNA(Tyr)(GTA) and an Arabidopsis intron-containing tRNA(Met)(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of beta-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA(Ser) gene and the GUS reporter gene resulted in approximately 10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA(Tyr) or tRNA(Met) genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA(Met)(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA(Tyr) to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA(Tyr) and tRNA(Met) were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.
Arabidopsis↗
New chromatographic and biochemical strategies for quick preparative isolation of tRNA.
A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.
Acylation↗
Sequence analysis of the tRNA(Tyr) and tRNA(Lys) genes and evidence for the transcription of a chloroplast-like tRNA(Met) in maize mitochondria.
The nucleotide sequences of three tRNA genes and their flanking regions from the maize mitochondrial genome is reported. These genes, which are located in the same region of the genome between the 14-kb inverted repeats, are transcribed in the mitochondria and code for tRNA(Lys) (anticodon UUU) tRNA(Met) (CAU) and tRNA(Tyr) (GUA). The very high homology shown by the tRNA(Met) gene with its chloroplast counterpart indicates that it probably originates from a chloroplast DNA insertion. The analysis of the upstream regions of these genes showed that the tRNA(Tyr) and the tRNA(Lys) genes possess the consensus sequence AAGAANRR, which could act as a promoter sequence in higher plant mitochondria.
Base Sequence↗
RNase P RNA in Candida glabrata mitochondria is transcribed with substrate tRNAs.
The biosynthesis of some mitochondrial enzymes requires contributions of both the mitochondrial and nuclear genomes. The ribonucleoprotein enzyme Ribonuclease P (RNase P) is composed of a mitochondrial encoded RNA and nuclear coded protein in many yeasts, including C. glabrata. We have determined that there are at least two sites of transcription initiation that contribute to the expression of the mitochondrial RNase P RNA. A nonanucleotide promoter sequence is located upstream of the initiator tRNA while the other site of initiation of transcription is at an undetermined upstream site. An analysis of the transcripts from the region of the RNase P gene demonstrates directly that the RNase P RNA is present in large primary transcripts and located between the precursors to the initiator tRNAf(Met) and tRNA(Pro) genes. Thus this enzyme subunit is synthesized with some of its substrate tRNAs. An activity with cleavage site specificity like a previously described endonuclease that cleaves near the 3' end of tRNAs, RNase P activity and one or more additional endonucleases or exonucleases not described previously are required to convert the primary transcript to its final functional RNAs.
Base Sequence↗
Mitochondrial DNA of Hydra attenuata (Cnidaria): a sequence that includes an end of one linear molecule and the genes for l-rRNA, tRNA(f-Met), tRNA(Trp), COII, and ATPase8.
The 3231-nucleotide-pair (ntp) sequence of one end of one of the two linear mitochondrial (mt) DNA molecules of Hydra attenuata (phylum Cnidaria, class Hydrozoa, order Anthomedusae) has been determined. This segment contains complete genes for tRNA(f-Met), l-rRNA, tRNA(Trp), subunit 2 of cytochrome c oxidase (COII), subunit 8 of ATP synthetase (ATPase8), and the 5' 136 ntp of ATPase6. These genes are arranged in the order given and are transcribed from the same strand of the molecule. As in two other cnidarians, the hexacorallian anthozoan Metridium senile and the octocorallian anthozoan Sarcophyton glaucum, the mt-genetic code of H. attenuata is near standard. The only modification appears to be that TGA specifies tryptophan rather than termination. Also as in M. senile and S. glaucum, the encoded H. attenuata mt-tRNA(f-Met) has primary and secondary structural features resembling those of Escherichia coli initiator tRNA(t-Met). As the encoded mt-tRNA(Trp) cannot be folded into a totally orthodox secondary structure, two alternative forms are suggested. The encoded H. attenuata mt-l-rRNA is 1738 nt, which is 451 nt shorter than the M. senile mt-l-rRNA. Comparisons of secondary structure models of these two mt-l-rRNAs indicate that most of the size difference results from loss of nucleotides in the H. attenuata molecule at a minimum of 46 locations, which includes elimination of six distinct helical elements.
Adenosine Triphosphatases↗
Specific RNA cleavages induced by manganese ions.
The specificity and efficiency of manganese ion-induced RNA hydrolysis was studied with several tRNA molecules. In case of yeast tRNA(Phe), the main cleavage occurs at p16 and minor cuts at p17-18, p20-21, p34 and p36-37. The major Mn(II)-induced cut in yeast elongator tRNA(Met) is also located in the D-loop at p16 and it is stronger than that observed in tRNA(Phe). In initiator tRNA(Met) from yeast two strong Mn(II) cleavages of equal intensity occur at p16 and p17. This is in contrast with single, much weaker cuts induced in the D-loop of that tRNA by Mg(II), Eu(III) and Pb(II) ions. Interestingly, in case of yeast tRNA(Glu) the main cleavage caused by Mn(II), Mg(II) and Pb(II) ions occurs in the anticodon loop. The involvement of hypermodified base mnm5s2U in this cleavage was ruled out based on results obtained with in vitro transcript of yeast tRNA(Glu) anticodon arm. Mutation of a single base A37G in the anticodon loop of the transcript drastically reduced the specificity of Mn(II)-induced hydrolysis.
Anticodon↗
Molecular mimicry in translational control of E. coli threonyl-tRNA synthetase gene. Competitive inhibition in tRNA aminoacylation and operator-repressor recognition switch using tRNA identity rules.
We previously showed that: (i) E.coli threonyl-tRNA synthetase (ThrRS) binds to the leader of its mRNA and represses translation by preventing ribosome binding to its loading site; (ii) the translational operator shares sequence and structure similarities with tRNA(Thr); (iii) it is possible to switch the specificity of the translational control from ThrRS to methionyl-tRNA synthetase (MetRS) by changing the CGU anticodon-like sequence to CAU, the tRNA(Met) anticodon. Here, we show that the wild type (CGU) and the mutated (CAU) operators act as competitive inhibitors of tRNA(Thr) and tRNA(fMet) for aminoacylation catalyzed by E.coli ThrRS and MetRS, respectively. The apparent Kd of the MetRS/CAU operator complex is one order magnitude higher than that of the ThrRS/CGU operator complex. Although ThrRS and MetRS shield the anticodon- and acceptor-like domains of their respective operators, the relative contribution of these two domains differs significantly. As in the threonine system, the interaction of MetRS with the CAU operator occludes ribosome binding to its loading site. The present data demonstrate that the anticodon-like sequence is one major determinant for the identity of the operator and the regulation specificity. It further shows that the tRNA-like operator obeys to tRNA identity rules.
Acylation↗
Ribosome modulation factor: stationary growth phase-specific inhibitor of ribosome functions from Escherichia coli.
Ribosome modulation factor (RMF) is an Escherichia coli protein associated with 100S ribosome dimers, which are formed at stationary growth phase or in slowly growing cells. RMF either purified from stationary-phase ribosomes or synthesized by a chemical method was examined for its functions. By adding either natural or synthetic RMF to 70S ribosomes prepared from both exponentially growing and stationary phase cells, 100S ribosome dimers were generated in a concentration-dependent manner. Protein synthesis in vitro was inhibited concomittantly with the formation of 100S ribosomes. The binding of aminoacyl-tRNA to ribosomes was inhibited in parallel. Taken together we propose that RMF is a stationary phase-specific inhibitor of ribosome functions and 100S dimers are stored forms of ribosomes.
Amino Acid Sequence↗
Translation initiation complex formation with 30 S ribosomal particles mutated at conserved positions in the 3'-minor domain of 16 S RNA.
Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting. These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex. Initiation factor-3-dependent selection of tRNA(fMet) from a mixture of tRNA(fMet) and tRNA(Phe) occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants. Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA(fMet) and tRNA(Phe) for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA(fMet). The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m2(6)A, in the synthetic RNA. Synthetic particles containing the mutation U1512C, which converts the universal U.G pair to C.G enhanced both tRNA(fMet) binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect. Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3'-minor domain, also enhanced tRNA(fMet) selectivity, in this case by reducing complex formation with elongator tRNA. Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels. The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence. This result had not been detected by standard tRNA-30 S binding assays. Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA(fMet).
Base Sequence↗
The determination of the in situ structure by nuclear spin contrast variation.
Polarized neutron scattering from polarized nuclear spins in hydrogenous substances opens a new way of contrast variation. The enhanced contrast due to proton spin polarization was used for the in situ structure determination of tRNA of the functional complex of the E. coli ribosome.
Crystallography↗
Transport of intracellularly active ribozymes to the cytoplasm.
Ribozymes are RNA molecules with enzymatic activity which selectively bind and cleave specific target RNAs. To date, numerous studies directed toward the application of ribozymes in vivo have been performed and many successful experiments have been reported. However, to induce high-level activities of ribozymes in vivo, several factors must be considered. Here we report that the cytoplasmic localization of ribozymes is important for their intracellular activity in mammalian cells. Northern blot analysis revealed that a tRNA(Val) ribozyme, which can assume a cloverleaf structure similar to that of a native tRNA, is efficiently transported to the cytoplasm. In contrast, the tRNAiMet-driven ribozyme, which does not maintain the cloverleaf structure, remained predominantly in the nucleus. In correlation with the localization, the activity of the exported ribozyme was higher than that of the ribozyme retained in the nucleus. These results should provide insight into the design of ribozymes that have high-level activity in mammalian cells.
Base Sequence↗
Stronger affinity of reticulocyte release factor than natural suppressor tRNASer for the opal termination codon.
Animal natural suppressor tRNA did not affect the release reaction of reticulocyte release factor (RF) at the same concentration of tRNA (both estimated as being present at a similar level of 3-5 X 10(-8) M in vivo); even at a 10-fold greater concentration the tRNA did not prevent the release reaction with RF. In order to confirm this result, the Ka values were determined. The Ka value between RF and UGA was 1.26 X 10(6) M-1 and that between the suppressor tRNA and UGA amounted to 8 X 10(3) M-1. This result showed that RF had a 150-fold stronger affinity than suppressor tRNA for the opal termination codon. Incorporation of phosphoserine into phosphoprotein via phosphoseryl-tRNA was inhibited by addition of RF to the reaction mixture. These results suggest that animal natural suppressor tRNA in the normal state does not perform its suppressor function, except in special cases where mRNA has the context structure near the opal termination codon (UGA).
Animals↗
Differences in 23 S rRNA-protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex. Probing by bifunctional Pt(II)-containing reagent.
rRNA-protein cross-links in free E. coli 35S-labeled 70 S ribosomes and in the initiation complex 35S-labeled 70 S ribosome.AUGU6.fMet-tRNA(fMet) were studied with the aid of a new type of binuclear Pt(II) compound - dichlorotetra-ammine(1,6-hexamethylenediaminediplatinum++ +) dichloride. The use of this reagent allowed us to reveal differences in the rRNA-protein neighbourhood in free 70 S ribosomes and in the initiation complex. Proteins L3, L6, L23 and L25 were shown to cross-link to 23 S rRNA only in the initiation complex, whereas proteins L1, L13, L14, L16, L17, L18, L22, L28 and S1 did so in both free ribosomes and the complex. 16 S rRNA was found to be cross-linked preferentially to a single protein, S1, in both states of the ribosomes.
Centrifugation, Density Gradient↗