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PubMed1993

The conformation of tRNA genes. Chemical modification studies.

It has been suggested that eukaryotic tRNA genes might adopt a higher order stem and loop structure to facilitate transcription by interaction of their variably spaced intragenic promoter blocks. Using sodium bisulphite, which reacts specifically with cytosine residues in single-stranded nucleic acids, no deamination of C in the TTCGAA sequence of the 3' ICR of a tRNA(Leu) gene could be detected under conditions which caused 60% deamination of cytosine residues within the loop region of a synthetic cruciform cloned in the same negatively supercoiled plasmid vector. We conclude that, under these conditions, such structures occur in tRNA genes very rarely, if at all.

DNA, Superhelical↗
PubMed2002

Isolation and characterization of the putative nuclear modifier gene MTO1 involved in the pathogenesis of deafness-associated mitochondrial 12 S rRNA A1555G mutation.

The human mitochondrial 12 S rRNA A1555G mutation has been found to be associated with aminoglycoside-induced and non-syndromic deafness. However, putative nuclear modifier gene(s) have been proposed to regulate the phenotypic expression of this mutation. In yeast, the mutant alleles of MTO1, encoding a mitochondrial protein, manifest respiratory-deficient phenotype only when coupled with the mitochondrial 15 S rRNA P(R)454 mutation corresponding to human A1555G mutation. This suggests that the MTO1-like modifier gene may influence the phenotypic expression of human A1555G mutation. Here we report the identification of full-length cDNA and elucidation of genomic organization of the human MTO1 homolog. Human Mto1 is an evolutionarily conserved protein that implicates a role in the mitochondrial tRNA modification. Functional conservation of this protein is supported by the observation that isolated human MTO1 cDNA can complement the respiratory deficient phenotype of yeast mto1 cells carrying P(R)454 mutation. MTO1 is ubiquitously expressed in various tissues, but with a markedly elevated expression in tissues of high metabolic rates including cochlea. These observations suggest that human MTO1 is a structural and functional homolog of yeast MTO1. Thus, it may play an important role in the pathogenesis of deafness-associated A1555G mutation in 12 S rRNA gene or mutations in tRNA genes.

Alleles↗
PubMed1995

1-Methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium.

1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg). A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C. The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA. Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu). Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent. The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs. 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT. Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected. We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog. Our results also suggest that tRNA modification may be a regulatory device for gene expression.

3-Isopropylmalate Dehydrogenase↗
PubMed1994

maf1 mutation alters the subcellular localization of the Mod5 protein in yeast.

Two forms of Mod5p, a tRNA modification enzyme, are found in three intracellular compartments, mitochondria, cytoplasm and nucleus, but are encoded by a single MOD5 gene. The two forms of the enzyme, Mod5p-I and Mod5p-II differ at the N-termini and are produced by initiation of translation at different start codons. Mod5p-I does contain a mitochondrial targeting signal and is distributed between mitochondria and cytoplasm, whereas Mod5p-II is found in the cytosol and nucleus (Boguta, M., et al. 1994, Mol. Cell. Biol. 14, 2298-2306). In the present work mutants which mislocalize the Mod5p-I enzyme were isolated. The screen was based on a correlation between the amount of cytosolic protein and the efficiency of tRNA mediated suppression. Identification of mutants is possible because a red pigment accumulates in the cells unable to suppress an ade2-1 nonsense allele. The maf1 mutant, with an altered intracellular localization of the Mod5p-I protein, was isolated. Immunofluorescence data suggest that the mutation causes mislocalization of the Mod5p-I to the nucleus.

Antimicrobial Cationic Peptides↗
PubMed2006

Dimerisation-dependent GTPase reaction of MnmE: how potassium acts as GTPase-activating element.

MnmE, a Guanine nucleotide-binding protein conserved between bacteria and man, is involved in the modification of tRNAs. Here we provide biochemical and X-ray structural evidence for a new GTP-hydrolysis mechanism, where the G-domains of MnmE dimerise in a potassium-dependent manner and induce GTP hydrolysis. The structure in the presence of GDP-AlFx and potassium shows how juxtaposition of the subunits induces a conformational change around the nucleotide which reorients the catalytic machinery. A critical glutamate is positioned such as to stabilise or activate the attacking water. Potassium provides a positive charge into the catalytic site in a position analogous to the arginine finger in the Ras-RasGAP system. Mutational studies show that potassium-dependent dimerisation and GTP hydrolysis can be uncoupled and that interaction between the G-domains is a prerequisite for subsequent phosphoryl transfer. We propose a model for the juxtaposition of G-domains in the full-length protein and how it induces conformational changes in the putative tRNA-modification centre.

Aluminum Compounds↗
PubMed2002

Enzymatic modification of tRNAs: MiaB is an iron-sulfur protein.

The product of the miaB gene, MiaB, from Escherichia coli participates in the methylthiolation of the adenosine 37 residue during modification of tRNAs that read codons beginning with uridine. A His-tagged version of MiaB has been overproduced and purified to homogeneity. Gel electrophoresis and size exclusion chromatography revealed that MiaB protein is a monomer. As isolated MiaB contains both iron and sulfide and an apoprotein form can chelate as much as 2.5-3 iron and 3-3.5 sulfur atoms per polypeptide chain. UV-visible and EPR spectroscopy of MiaB indicate the presence of a [4Fe-4S] cluster under reducing and anaerobic conditions, whereas [2Fe-2S] and [3Fe-4S] forms are generated under aerobic conditions. Preliminary site-directed mutagenesis studies suggest that Cys(157), Cys(161), and Cys(164) are involved in iron chelation and that the cluster is essential for activity. Together with the previously shown requirement of S-adenosylmethionine (AdoMet) for the methylthiolation reaction, the finding that MiaB is an iron-sulfur protein suggests that it belongs to a superfamily of enzymes that uses [Fe-S] centers and AdoMet to initiate radical catalysis. MiaB is the first and only tRNA modification enzyme known to contain an Fe-S cluster.

Chromatography, High Pressure Liquid↗
PubMed2002

Trm7p catalyses the formation of two 2'-O-methylriboses in yeast tRNA anticodon loop.

The genome of Saccharomyces cerevisiae encodes three close homologues of the Escherichia coli 2'-O-rRNA methyltransferase FtsJ/RrmJ, designated Trm7p, Spb1p and Mrm2p. We present evidence that Trm7p methylates the 2'-O-ribose of nucleotides at positions 32 and 34 of the tRNA anticodon loop, both in vivo and in vitro. In a trm7Delta strain, which is viable but grows slowly, translation is impaired, thus indicating that these tRNA modifications could be important for translation efficiency. We discuss the emergence of a family of three 2'-O-RNA methyltransferases in Eukaryota and one in Prokaryota from a common ancestor. We propose that each eukaryotic enzyme is located in a different cell compartment, in which it would methylate a different RNA that can adopt a very similar secondary structure.

Amino Acid Sequence↗
PubMed2001

The mere lack of rT modification in initiator tRNA does not facilitate formylation-independent initiation in Escherichia coli.

Formylation of initiator methionyl-tRNA is essential for normal growth of eubacteria. However, under special conditions, it has been possible to initiate protein synthesis with unformylated initiator tRNA even in eubacteria. Earlier studies suggested that the lack of ribothymidine (rT) modification in initiator tRNA may facilitate initiation in the absence of formylation. In this report we show, by using trmA strains of Escherichia coli (defective for rT modification) and a sensitive in vivo initiation assay system, that the lack of rT modification in the initiators is not sufficient to effect formylation-independent initiation of protein synthesis.

Acylation↗
PubMed2006

Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations.

Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.

Base Sequence↗
PubMed2007

Structural insights into the GTPase domain of Escherichia coli MnmE protein.

The Escherichia coli MnmE protein is a 50-kDa multidomain GTPase involved in tRNA modification. Its homologues in eukaryotes are crucial for mitochondrial respiration and, thus, it is thought that the human protein might be involved in mitochondrial diseases. Unlike Ras, MnmE shows a high intrinsic GTPase activity and requires effective GTP hydrolysis, and not simply GTP binding, to be functionally active. The isolated MnmE G-domain (165 residues) conserves the GTPase activity of the entire protein, suggesting that it contains the catalytic residues for GTP hydrolysis. To explore the GTP hydrolysis mechanism of MnmE, we analyzed the effect of low pH on binding and hydrolysis of GTP, as well as on the formation of a MnmE transition state mimic. GTP hydrolysis by MnmE, but not GTP binding or formation of a complex with mant-GDP and aluminium fluoride, is impaired at acidic pH, suggesting that the chemistry of the transition state mimic is different to that of the true transition state, and that some residue(s), critical for GTP hydrolysis, is severely affected by low pH. We use a nuclear magnetic resonance (NMR)-based approach to get insights into the MnmE structure and properties. The combined use of NMR restraints and homology structural information allowed the determination of the MnmE G-domain structure in its free form. Chemical shift structure-based prediction provided a good basis for structure refinement and validation. Our data support that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis, although Arg252 may play a role in stabilization of the transition state.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis reveals the wide-ranging effects of the novel, iron-responsive regulator RirA in Rhizobium leguminosarum bv. viciae.

The wide-ranging effects of RirA, a novel Fe-responsive regulator of gene expression in Rhizobium leguminosarum bv. viciae, were monitored on 2D gels. Approximately 100 proteins were expressed at higher levels in a RirA(-) mutant, compared to wild type. These included the products of the sufS(2)BCDS(1)XA operon, which probably specifies the synthesis of [FeS] clusters. Using lac fusions, this operon was confirmed to be regulated by RirA in response to Fe availability. Genes for some ABC transporters, and a protein that may be involved in making a phenazine-like molecule, were also repressed by Fe in a RirA-dependent way. Strikingly, at least 17 proteins were reduced in abundance in the RirA(-) mutant. These included three ABC transporters, a GatB-like enzyme involved in tRNA modification, and a protein that may confer bacteriocin resistance. As judged by lac reporter fusions, this apparently positive control by RirA was probably due to post-transcriptional effects, in at least some cases. Therefore, although RirA shows no sequence similarity to Fur or DtxR, it functions as a wide-ranging, Fe-responsive regulator.

Bacterial Proteins↗
PubMed1989

A rapid diethylaminoethyl paper disk assay for transfer RNA sulfurtransferase.

A rapid assay for tRNA sulfurtransferase from Escherichia coli was developed, reducing the time needed to determine enzyme activity from 11 to 2 h. The reaction measured is the transfer of sulfur from [35S]cysteine to acceptor sites in a thionucleotide-deficient tRNA substrate. Processing is done by binding the product, [35S]-tRNA, to DEAE-cellulose filter disks. The disks are then treated to remove unreacted [35S]cysteine, cysteine-protein adducts and [35S]cysteinyl-tRNA. The DE81 disk assay and the 11-h standard assay are shown to give identical values over a wide range of incubation times and enzyme levels. Incorporation was greater when thionucleotide-deficient tRNA was used as substrate, as compared to fully modified tRNA. [35S]-tRNA was found to be the major reaction product, although some [35S]cysteine was also bound to the filters. The major thionucleoside labeled in nucleoside digests was 4-thiouridine, as determined by Bio-Gel P2 chromatography. We also observed other labeled peaks by this method, in amounts too small for positive identification. This rapid assay should be useful in the purification and study of this uncharacterized class of tRNA modification enzymes.

Escherichia coli↗
PubMed1992

Protein synthesis in HL-60 cells treated with DMSO and hypoxanthine.

Short-term treatment of the HL-60 cells with DMSO and hypoxanthine, inducers of granulocytic differentiation, was reported to cause a rapid increase in protein synthesis. This effect was ascribed to the insertion of inosine in the wobble position of the tRNA anticodon and consequently increasing codon recognition potential. In this study we have re-investigated the effects of DMSO and/or hypoxanthine on protein synthesis. In contrast to their findings we were unable to demonstrate stimulated protein synthesis in either short- or long-term treatment with these agents. Polysome analysis under these conditions revealed that polysomes were disaggregated. Finally, the activity of tRNA-hypoxanthine ribosyltransferase, an enzyme responsible for the insertion of inosine in the anticodon, was also relatively low. Under these circumstances, we propose that tRNA modification is not essential in the regulation of protein synthesis.

Cells, Cultured↗
PubMed1991

Evidence for demand-regulation of ribosome accumulation in E coli.

We have determined the relative concentrations of ribosomes accumulated under different growth conditions for a number of translational mutants as well as for some natural isolates of Escherichia coli. The mutants are a tRNA modification mutant (miaA), a streptomycin resistant (SmR) and a streptomycin pseudodependent (SmP) mutant as well as two ribosome ambiguity (ram) mutants. The natural isolates used in this study are known to function with submaximal ribosome kinetics. The data show that for all the ribosome mutants the concentration of ribosomes relative to that in wild type bacteria increases when the growth rate decreases. A small increase is also seen in the natural isolates. In contrast, the miaA mutant shows no increase in ribosome concentration under the same slow growth conditions. The results suggest that bacteria with kinetically impaired ribosomes can to some extent increase the number of ribosomes accumulated under poor growth conditions in order to compensate for their slower function. We use this observation to explain in part how bacteria growing in natural environments can escape the strong selection for maximized growth rates and for optimized ribosomes that are characteristic of laboratory strains.

Culture Media↗
PubMed2006

S-Adenosylmethionine in protozoan parasites: functions, synthesis and regulation.

S-adenosylmethionine is one of the most frequently used enzymatic substrates in all living organisms. It plays a role in all biological methyl transfer reactions in as much as it is a donor of propylamine groups in the synthesis of the polyamines spermidine and spermine, it participates in the trans-sulphuration pathway to cysteine one of the three amino acids involved in glutathione and trypanothione synthesis in trypanosomatids and finally it is a source of the 5-deoxyadenosyl radicals, which are involved in many reductive metabolic processes, biodegradative pathways, tRNA modification and DNA repair. This mini-review is an update of the progress on the S-adenosylmethionine synthesis in different representative protozoan parasites responsible for many of the most devastating so-called tropical diseases that have an enormous impact on global health.

Animals↗
PubMed1985

Identification of the site of cross-linking in 16S rRNA of an aromatic azide photoaffinity probe attached to the 5'-anticodon base of A site bound tRNA.

The site of Escherichia coli 16S ribosomal RNA cross-linked to the 5'-anticodon base of A site bound E. coli valyl-tRNA was identified. Cross-linking was via the affinity probe 6-[(2-nitro-4-azidophenyl)amino]caproate (NAK) or 3-[[2-[(2-nitro-4-azidophenyl)amino]ethyl]dithio]propionate (SNAP) attached to the carboxyl group of the 5'-anticodon base 5-(carboxyethoxy)uridine via an ethylenediamine spacer [Gornicki, P., Ciesiolka, J., & Ofengand, J. (1985) Biochemistry (preceding paper in this issue)]. With both probes, RNase T1 digestion of the isolated 16S RNA-tRNA covalent complex, 5'-32P postlabeling, and gel electrophoresis yielded two oligonucleotides larger than any fragments from non-cross-linked tRNA or rRNA. Appearance of the oligomers was dependent on the presence of the probe on the tRNA. Unmodified tRNA in the A and/or P sites did not yield any product. The presence of elongation factor Tu in the incubation mixture was also required. Dithiothreitol (DDT) treatment of the SNAP-induced covalent complex prior to electrophoresis also abolished the oligomers. Only the larger of the two oligomers (present in a 3:1 ratio) was sequenced. The SNAP dimer was cleaved with DTT, and the rRNA and tRNA oligomers were separated and sequenced as monomers. The NAK dimer was sequenced without cleavage by taking advantage of the differences in electrophoretic mobility among sequence and/or composition isomers of the same length. In both cases, the rRNA oligomer was identified as UACACACCG1401, and the nucleotide cross-linked was shown to be the C1400 residue. The expected tRNA modification site was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1981

High-resolution phosphorus nuclear magnetic resonance spectra of yeast phenylalanine transfer ribonucleic acid. Metal ion effects and tentative partial assignment of signals.

The temperature and metal ion dependence of the 31P NMR spectra of yeast phenylalanine tRNA is presented, and a tentative assignment of some of the individual phosphate signals is offered. Signals A, C, F, P, and T have been assigned to the magnesium binding site of phosphates by monitoring the magnesium ion dependence in the 31P NMR spectra. In the presence of 10 mM Mg2+, specific paramagnetic line broadening effects are observed for signals A, D, E, and U upon addition of 0.001-0.1 Mn2+ ion per tRNA molecule. Through the combination of the above metal in experiments, X-ray studies, and earlier 1H and 31P NMR studies [particularly the tRNA modification 31P NMR experiments of Salemink et al. [Salemink, P..J.M., Swarthof T., & Hilbers, C.W. (1979) Biochemistry 18, 3477]], an initial assignment of some of these signals is attempted.

Base Sequence↗
PubMed2007

Natural expansion of the genetic code.

At the time of its discovery four decades ago, the genetic code was viewed as the result of a "frozen accident." Our current knowledge of the translation process and of the detailed structure of its components highlights the roles of RNA structure (in mRNA and tRNA), RNA modification (in tRNA), and aminoacyl-tRNA synthetase diversity in the evolution of the genetic code. The diverse assortment of codon reassignments present in subcellular organelles and organisms of distinct lineages has 'thawed' the concept of a universal immutable code; it may not be accidental that out of more than 140 amino acids found in natural proteins, only two (selenocysteine and pyrrolysine) are known to have been added to the standard 20-member amino acid alphabet. The existence of phosphoseryl-tRNA (in the form of tRNACys and tRNASec) may presage the discovery of other cotranslationally inserted modified amino acids.

Amino Acyl-tRNA Synthetases↗