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PubMed1991

Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA.

We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.

Electrophoresis, Agar Gel↗
PubMed1991

Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae. I. Purification and physical properties.

Compared to other tRNAs all known histidine tRNAs have the unique feature of possessing an additional nucleotide at their 5' end. It is usually a guanosine residue but not in bacteriophage T5 tRNA which carries an additional uridine. The additional nucleotide is not encoded in eukaryotic histidine tRNA genes but is added in a post-transcriptional modification reaction (Cooley, L., Appel, B., and Söll, D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6475-6479) by histidine tRNA guanylyltransferase (tRNAHis guanylyltransferase). Here we report the purification of this enzyme from Saccharomyces cerevisiae and the determination of some of its physical properties. Six different steps including Polymin P precipitation, chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, ATP-agarose, and gel filtration on Superose 12 were employed for the purification of the guanylyltransferase from an S-100 extract. A Stokes radius of 46.5 +/- 0.5 A and a sedimentation coefficient (S20,w) of 7.8 +/- 0.2 were determined by gel filtration and rate zonal sedimentation, respectively. A relative molecular weight (Mr) of approximately 120,000 was calculated for the purified native enzyme. The Mr of the denatured protein is approximately 58,000 as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate a homodimeric (alpha 2) structure for the enzyme. Among all acceptor RNAs in unfractionated tRNA only tRNAHis is a substrate for the purified guanylyltransferase. The reaction requires ATP, a guanosine substrate, and a divalent metal ion. Treatment of guanylyltransferase with 5,5'-dithiobis(2-nitrobenzoic)-acid abolishes activity; this suggests the importance of sulfhydryl groups for enzymatic activity. The enzyme shows discrimination among different histidine tRNA species; tRNA from plant and prokaryotes are better substrates than mammalian and insect tRNAs.

Adenosine Triphosphate↗
PubMed2005

Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases.

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.

Base Sequence↗
PubMed1990

Relaxation of a transfer RNA specificity by removal of modified nucleotides.

The molecular recognition of specific transfer RNAs by the appropriate aminoacyl-tRNA synthetase is an important step in determining the accuracy of translation of the genetic message from nucleic acids into proteins. Recent studies using variant tRNAs with specific sequence modifications have indicated particular regions that determine their identity. Here we consider whether the base modifications commonly found in tRNAs contribute to their identity. Although unmodified tRNA(Asp) is charged with aspartate as efficiently as the modified native tRNA, it is mischarged with arginine with considerably increased efficiency. Our results indicate that post-transcriptional modification of tRNAs introduces structural 'anti-determinants', restricting the efficiency with which the tRNAs are charged with inappropriate amino acids.

Arginine↗
PubMed1992

Heavy-atom refinement against solvent-flattened phases.

A new algorithm for refinement of heavy-atom parameters is defined by an iterative procedure where external phases are provided by density modification. This algorithm is applied to two cases, tRNA(Asp) and the complex between tRNA(Asp) and aspartyl-tRNA synthetase. In the first case, where the structure was solved by multiple isomorphous replacement (MIR) methods, it was found that the new method gives accurate values for the native-derivative scale and four occupancy of heavy-atom sites. Position refinement was more delicate and it needed to be handled in a restricted resolution range. In the second case, where a similar method was used in the early stages of solving the phase problem, it slightly decreased the phase error. It was followed by an improvement of the density-modification masks, which led to better maps at higher resolution.

Algorithms↗
PubMed2004

Mitochondria-specific RNA-modifying enzymes responsible for the biosynthesis of the wobble base in mitochondrial tRNAs. Implications for the molecular pathogenesis of human mitochondrial diseases.

Human mitochondrial (mt) tRNA(Lys) has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (taum5s2U), at its anticodon wobble position. We previously found that the mt tRNA(Lys), carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the taum5s2U modification. Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity. Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed. Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding. Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF.

Amino Acid Sequence↗
PubMed1998

Comparison of tRNA conformation during different phases of reproduction.

The present study is a comparison of tRNA conformation from ovary of Heteropneustes fossilis in its active phase of reproduction (when it is highly engaged in protein synthesis i.e. previtellogenic phase) with inactive phase (when tRNA is mainly stored in mature ovary i.e. spawning phase). Transfer RNA of active phase is shown to be compact, flexible and susceptible towards nuclease. Compact tRNA structure is evidenced by higher hyperchromicity and presence of relatively less Gm modifications thereby allowing adequate hydrogen bonding between D loop and T loop. Higher sensitivity of tRNA towards Mg++ reflects its higher flexibility towards internal environment. This structure of tRNA may be required for active protein synthesis. On the other hand tRNA of inactive phase is shown to be relaxed but resistant towards nuclease which may be favoured for storage in mature ova of a teleost as maternal carry over.

Animals↗
PubMed1978

Studies on transfer RNA from mycobacteria.

Active preparations of tRNA and aminoacyl-tRNA synthetases have been isolated from exponentially growing cells of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. Though the aminoacyl-tRNA synthetases of older cells retain their activity, the tRNAs seem to undergo modification and show poorer activity. The mycobacterial enzyme preparations catalyse homologous and heterologous aminoacylation between tRNA from the two species (M. smegmatis and M. tuberculosis H37Rv) or from Escherichia coli, with equal efficiency; tRNA samples from eukaryotic cells (yeast and rat liver) do not serve as substrates for the mycobacterial synthetases. The analytical separation of the different amino acid specific tRNAs from M. smegmatis resembles the pattern found in other bacteria. Purification of valine- (three species) and methionine-specific tRNA (two species) to 70-80% purity has been accomplished by using column-chromatographic techniques. Of the two species of tRNAMet, one can be formylated in the presence of formyl tetrahydrofolate and the transformylase from mycobacteria.

Amino Acyl-tRNA Synthetases↗
PubMed2000

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.

Acylation↗
PubMed2001

Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses.

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.

Anticodon↗
PubMed1990

Interactions with tRNA(Lys) induce important structural changes in human immunodeficiency virus reverse transcriptase.

Retroviral RNA-dependent DNA polymerase (reverse transcriptase or RT) uses the 3'OH end of a cellular tRNA as primer to initiate DNA synthesis. Previous work with avian retrovirus has shown that reverse transcriptase is implicated in the selection of cellular virion-encapsidated tRNAs and has shown that the primer tRNA is positioned on the primer binding site near the 5' end of the viral RNA. These mechanisms support the idea that the retroviral polymerase should form complexes with primer tRNA and the specific encapsidated ones. The genomic sequence of human immunodeficiency virus (HIV) allows the prediction that tRNA(Lys3) is the natural primer. In this article we show, using the mobility shift assay, that recombinant HIV reverse transcriptase is able to form a complex with bovine tRNA(Lys.) By fluorescence studies and alpha-chymotrypsin analysis we have observed a modification of the enzyme conformation when reverse transcriptase is bound to the putative primer tRNA. This structural change is specific for tRNA(Lys) although the retroviral polymerase is able to interact with other tRNAs.

HIV↗
PubMed2004

Crystal structure of KD93, a novel protein expressed in human hematopoietic stem/progenitor cells.

The crystal structure of a novel hypothetical protein, KD93, expressed in human hematopoietic stem/progenitor cells, was determined at 1.9A resolution using the multiple-wavelength anomalous dispersion (MAD) method. The protein KD93, which is encoded by the open reading frame HSPC031, is a NIP7 homologue and belongs to the UPF0113 family. The structural and functional information for the group of homologues has not yet been determined. Crystallographic analysis revealed that the overall fold of KD93 consists of two interlinked alpha/beta domains. Structure-based homology analysis with DALI revealed that the C domain of KD93 matches the PUA domain of some RNA modification enzymes, especially that of archaeosine tRNA-ribosyltransferase (ArcTGT), which suggests that its possible molecular function is related to RNA binding. The difference between the RNA binding regions of KD93 and ArcTGT in amino acid constitution and surface electrostatic potential indicate that they may have different RNA binding modes. The N domain of KD93 is a unique structure with no obvious similarity to other proteins with known three-dimensional structures. The high-resolution structure of KD93 provides a first view of a member of the family of hypothetical proteins. And the structure provides a framework to deduce and assay the molecular function of other proteins of the UPF0113 family.

Crystallography, X-Ray↗
PubMed1999

Transfer RNA modification: influence on translational frameshifting and metabolism.

Transfer RNA modification improves the rate of aa-tRNA selection at the A-site and the fitness in the P-site and thereby prevents frameshifting according to a new model how frameshifting occurs [Qian et al. (1998) Mol. Cell 1, 471-482]. Evidence that the presence of various modified nucleosides in tRNA also influences central metabolism, thiamine metabolism, and bacterial virulence is reviewed.

Frameshifting, Ribosomal↗
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↗
PubMed2000

Evolution of RNA editing in trypanosome mitochondria.

Two different RNA editing systems have been described in the kinetoplast-mitochondrion of trypanosomatid protists. The first involves the precise insertion and deletion of U residues mostly within the coding regions of maxicircle-encoded mRNAs to produce open reading frames. This editing is mediated by short overlapping complementary guide RNAs encoded in both the maxicircle and the minicircle molecules and involves a series of enzymatic cleavage-ligation steps. The second editing system is a C(34) to U(34) modification in the anticodon of the imported tRNA(Trp), thereby permitting the decoding of the UGA stop codon as tryptophan. U-insertion editing probably originated in an ancestor of the kinetoplastid lineage and appears to have evolved in some cases by the replacement of the original pan-edited cryptogene with a partially edited cDNA. The driving force for the evolutionary fixation of these retroposition events was postulated to be the stochastic loss of entire minicircle sequence classes and their encoded guide RNAs upon segregation of the single kinetoplast DNA network into daughter cells at cell division. A large plasticity in the relative abundance of minicircle sequence classes has been observed during cell culture in the laboratory. Computer simulations provide theoretical evidence for this plasticity if a random distribution and segregation model of minicircles is assumed. The possible evolutionary relationship of the C to U and U-insertion editing systems is discussed.

Animals↗
PubMed1972

RNA metabolism in HeLa cells at reduced temperature. II. Steps in the processing of transfer RNA.

Incubation of HeLa cells at 24 degrees C results in the modification of the processing of pre-tRNA to tRNA. Both methylation and size reduction were shown to take place in vitro when purified pre-tRNA was subjected to processing in a cytoplasmic extract of HeLa cells The migration of pre-tRNA from the nucleus to the cytoplasm was not significantly altered at 24 degrees C

Cell Fractionation↗
PubMed2003

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Anti-Bacterial Agents↗
PubMed1982

Dihydrouridine-deficient tRNAs in Saccharomyces cerevisiae.

A mutation in Saccharomyces cerevisiae, designated mia, is responsible for the production of isoaccepting tRNA molecules with reduced extents of nucleoside modifications. The mia isoacceptors of tRNAPhe and one of the mutant isoacceptors of tRNATyr were highly purified for nucleoside composition analyses. The data indicate that the mutant isoacceptors are lacking some of the dihydrouridine moieties. This is consistent with our previous hypothesis that the mutant isoacceptors were accumulated due to a defect in a modification process [Lo, R.Y.C. and Bell, J.B. (1981) Current Genetics 3, 73-82). Data from in vitro poly-U translation experiments also support the previous results, suggesting in vivo biological activity of these mutant tRNAs.

Base Composition↗