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PubMed1985

Antisuppressor mutation in Escherichia coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine.

Mutations in three Escherichia coli K-12 genes were isolated that reduce the efficiency of the lysine-inserting nonsense suppressor supL. These antisuppressor mutations asuD, asuE, and asuF map at 61.9, 25.3, and 76.3 min, respectively, on the E. coli chromosome. Biochemical and genetic analysis of the mutant strains revealed the reason for the antisuppressor phenotype for two of these genes. The activity of lysyl-tRNA synthetase was reduced in strains with asuD mutations. The modification of 5-methylaminomethyl-2-thiouridine, the wobble base of tRNALys, was impaired in asuE mutant strains, presumably at the 2-thiolation step.

Chromosome Mapping↗
PubMed2005

Modification at position 9 with 1-methyladenosine is crucial for structure and function of nematode mitochondrial tRNAs lacking the entire T-arm.

The mitochondria of the nematode Ascaris suum have tRNAs with unusual secondary structures that lack either the T-arm or D-arm found in most other organisms. Of the twenty-two tRNA species present in the mitochondria of A.suum, twenty lack the entire T-arm and two serine tRNAs lack the D-arm. To understand how such unusual tRNAs work in the nematode mitochondrial translation system, we analyzed post-transcriptional modifications of 11 mitochondrial tRNA species purified from A.suum, 10 of which lacked a T-arm and one of which lacked a D-arm. The most characteristic feature of nematode mitochondrial tRNAs lacking a T-arm was the presence of 1-methyladenosine at position 9 (m1A9). Synthesis of T-armless tRNAs with or without the modified nucleoside showed that T-armless tRNAs without the modification had much lower aminoacylation and EF-Tu-binding activities than native tRNAs. The addition of a single methyl group to A9 of these tRNAs was sufficient to restore nearly native levels of aminoacylation and EF-Tu-binding activity as well as tertiary structure, suggesting that m1A9 is a key residue for the activity of T-armless tRNAs. Thus, m1A9 is indispensable for the structure and function of T-armless tRNAs of nematode mitochondrial origin.

Adenosine↗
PubMed1984

Specific lack of the hypermodified nucleoside, queuosine, in hepatoma mitochondrial aspartate transfer RNA and its possible biological significance.

Tumor nucleic acids have frequently been found to be deficient in methylated and other modified nucleotides. In particular, cytoplasmic transfer RNAs (tRNAs) from various neoplasms partially lack the hypermodified nucleoside queuosine, a modification specific for anticodons of histidine-, tyrosine-, asparagine-, and aspartic acid-accepting tRNAs. Using aspartate tRNA as an example, we show here that liver mitochondria contain tRNA fully modified with respect to queuosine, while the corresponding tRNA from mitochondria of Morris hepatoma 5123D completely lacks this constituent. The sequences of these tRNAs, which were determined by a highly sensitive 32P-postlabeling procedure entailing the direct identification of each position of the polynucleotide chains, were found to be (sequence in text) Lack of queuosine in the hepatoma mitochondrial tRNA may be due to the inavailability of queuine in the hepatoma mitochondria for incorporation into tRNA or to inhibition of the modifying enzyme, tRNA (guanine)-transglycosylase, in the tumor. Taking into account results of others indicating a possible involvement of the queuosine modification in differentiation of eukaryotic cells, we hypothesize that the queuosine defect may develop at an early stage of carcinogenesis (i.e., during the promotion phase) and be directly involved in abnormalities of mitochondria which have been observed frequently in transformed cells and tumors.

Animals↗
PubMed2002

RNA editing by adenosine deaminases generates RNA and protein diversity.

RNA editing is defined as a post-transcriptional change of a gene-encoded sequence at the RNA level, excluding alterations due to processes such as pre-mRNA splicing and 3'-end formation. RNA editing is found in many organisms and can occur either by the insertion or deletion of nucleotides or by the substitution of bases by modification. The nucleoside inosine (I) was first detected in cytoplasmic tRNA and was later found in messenger RNA precursors (pre-mRNAs) and in viral transcripts. It is formed by hydrolytic deamination of a genomically encoded adenosine (A) at C6 of the base and this reaction is catalysed by a family of related enzymes. ADARs (for adenosine deaminases acting on RNA) catalyse A to I conversion either promiscuously or site-specifically in pre-mRNAs, viral RNAs and synthetic double-stranded RNAs (dsRNAs), whereas ADATs (for adenosine deaminases acting on tRNA) are involved in inosine formation in tRNAs. ADAT1 generates I at position 37 (3' of the anticodon) in eukaryotic tRNA(Ala). ADAT2 and ADAT3 function as a heterodimer which catalyses inosine formation at the wobble position (position 34) in eukaryotic tRNAs. Here, we review the state of knowledge on ADARs and ADATs and their RNA substrates, with an emphasis on the developments over the past few years that have increased the understanding of the mechanism of action of these enzymes and of the functional consequences of the widespread modification they catalyse.

Adenosine↗
PubMed2003

An RNA-modifying enzyme that governs both the codon and amino acid specificities of isoleucine tRNA.

The AUA codon-specific isoleucine tRNA (tRNA(Ile)) in eubacteria has the posttranscriptionally modified nucleoside lysidine (L) at the wobble position of the anticodon (position 34). This modification is a lysine-containing cytidine derivative that converts both the codon specificity of tRNA(Ile) from AUG to AUA and its amino acid specificity from methionine to isoleucine. We identified an essential gene (tilS; tRNA(Ile)-lysidine synthetase) that is responsible for lysidine formation in both Bacillus subtilis and Escherichia coli. The recombinant enzyme complexed specifically with tRNA(Ile) and synthesized L by utilizing ATP and lysine as substrates. The lysidine synthesis of this enzyme was shown to directly convert the amino acid specificity of tRNA(Ile) from methionine to isoleucine in vitro. Partial inactivation of tilS in vivo resulted in an AUA codon-dependent translational defect, which supports the notion that TilS is an RNA-modifying enzyme that plays a critical role in the accurate decoding of genetic information.

Amino Acid Sequence↗
PubMed1998

Localization of the binding site for the 3'-terminal sequence of tRNAPhe in subunits of phenylalanyl-tRNA synthetase from Thermus thermophilus.

A photoreactive tRNAPhe derivative containing a 4-thiouridine residue at the 3'-end (tRNAPhe-s4U-75) was prepared by tRNA nucleotidyltransferase-mediated incorporation of s4UMP into a tRNAPhe transcript lacking the 3'-terminal dinucleotide. The resulting tRNAPhe-s4U-75 was covalently bound to phenylalanyl-tRNA synthetase from Thermus thermophilus, and all criteria of an affinity modification were met. The main products of modification displaying various electrophoretic mobilities were formed by binding tRNAPhe-s4U-75 to the beta-subunit (major) of the enzyme. These data suggest that the nucleotide found at position 75 of tRNAPhe interacts with the beta-subunit of phenylalanyl-tRNA synthetase.

Base Sequence↗
PubMed2001

Specific N-terminal biotinylation of a protein in vitro by a chemically modified tRNA(fmet) can support the native activity of the translated protein.

Biotinylation of a protein generally involves chemical modification of a translated protein. Using this methodology, however, biotinylation at a specific position remains difficult. We investigated whether it would be possible to use an Escherichia coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group to introduce a biotin tag specifically at the N terminus. We report here that a biotin tag could be incorporated into the green fluorescent protein (GFP) at the N-terminal site, in the presence of an E. coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group. The biotinylated GFP was purified by simple monomeric streptavidin-agarose affinity column chromatography. Based on the total amount of GFP molecules, the purification yield and the biotin labelling efficiency of this system were approximately 7% and 10-20%, respectively, according to the densitometric analysis of Western blots. Judging from the results of a fluorescence imaging experiment, almost all the purified GFP molecules retained the native fluorescence activity. Importantly, the present results support the hypothesis that the E. coli initiator tRNA(fmet) aminoacylated with a relatively large substituent can be recognized by an E. coli ribosome and adequately placed at the P site to initiate translation.

Journal Article↗
PubMed1978

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.

Adenosine Triphosphate↗
PubMed1994

Proposed mechanisms for the involvement of lactoferrin in the hydrolysis of nucleic acids.

Lactoferrin has recently been proposed to have ribonuclease activity in the absence of bound iron. We and others have demonstrated previously that lactoferrin interacts with DNA and will bind a number of transition metal ions via surface-exposed histidyl residues. In the present study, we investigated the possibility that surface-bound copper ions on lactoferrin may catalyze the production of active oxygen species responsible for the hydrolysis of nucleic acids. Purified lactoferrin (apo- and holo-forms) was incubated with CuCl2 in solution to obtain lactoferrin with surface binding sites saturated by Cu(II)ions. the lactoferrin-Cu(II) complex was purified by Bio-Gel P-6 chromatography columns and tested for hydrolytic activity against DNA and RNA as analyzed by agarose gel electrophoresis. Incubation of lactoferrin-Cu(II) complexes with supercoiled plasmid Bluescript II SK DNA led to the rapid formation of relaxed open circular or linear forms of DNA characterized by changed electrophoretic mobility. Lactoferrin with bound Cu(II) also caused extensive degradation of yeast tRNA molecules in the presence of hydrogen peroxide. Covalent modification of surface-exposed histidyl residues by carboxyethylation with diethylpyrocarbonate abolished the lactoferrin-associated hydrolytic activity. These results indicate that lactoferrin-bound Cu(II) can indeed facilitate the hydrolysis of DNA and RNA molecules. Copper-binding sites on lactoferrin appear to serve as centers for repeated production of hydroxyl radicals via a Fenton-type Haber-Weiss reaction. Enhanced nuclease activity associated with elevated local concentrations of lactoferrin would promote microbial degradation.

Apoproteins↗
PubMed1980

A double base change in alternate base pairs induced by ultraviolet irradiation in a glycine transfer RNA gene.

The glyUsuAGA mutation affects Escherichia coli tRNA Gl y GGG, changing it to an AGA missense suppressor tRNA. Sequence studies have shown that the mutation involves a double base subsitution at the first and third positions of the tRNA anticodon, the result being a change in the anticodon from CCC to UCU. A system has been developed to facilitate the detection of this novel mutation, and we have shown that ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are effective in causing the double base change. A single observation of the mutation occurring spontaneously has been made also. The frequency of MNNG-induced glyUsuAGA mutations is compatible with their being caused by two separate mutagenic events. The frequency of UV-induced glyAGA mutations, however, strongly suggests that the occurrence of one base substitution strongly enhances the chance of finding the second substitution at the alternate position. In addition to the double change in the anticodon, the glyUsuAGA tRNA differs from tRNA Gl y GGG in that it bears a modification of the A adjacent to the 3' position of the anticodon. Most likely, this modified base is N-[9-(beta-D-ribofuranosyl)-purin-6-ylcarbamoyl] threonine.

Base Composition↗
PubMed1986

Mosquito mitochondrial transfer RNAs for valine, glycine and glutamate: RNA and gene sequences and vicinal genome organization.

We report the sequences of 3 transfer RNAs from mosquito (Aedes albopictus) mitochondria, those for valine (anticodon UAC), glutamic acid (anticodon UUC) and glycine (anticodon UCC), as well as sequences for the corresponding genes and for some neighboring mitochondrial genes. TRNAval is notable for its high level of psi, tRNAglu for its low level of G and C, and tRNAgly is notable in that it appears as two species widely separated in gel electrophoresis, differing only in modification status. TRNAglu is the first sequenced insect mitochondrial tRNA that would be expected to engage in U.R wobble (where U is a modified U in the first position of the anticodon, and R is G or A in the third position of codons), if the insect system followed the modified wobble rules proposed for mammalian and fungal mitochondria; and the sequence determined does fit the proposal. The gene for tRNAval follows immediately that for 12S ribosomal RNA. The gene for tRNAglu occurs in a cluster of 6 tRNA genes that is separated from the gene for tRNAgly by a short reading frame. Features of the DNA sequences are discussed with reference to Drosophila, and mammalian, mitochondrial genome organization.

Aedes↗
PubMed2006

Accurate energies of hydrogen bonded nucleic acid base pairs and triplets in tRNA tertiary interactions.

Tertiary interactions are crucial in maintaining the tRNA structure and functionality. We used a combined sequence analysis and quantum mechanics approach to calculate accurate energies of the most frequent tRNA tertiary base pairing interactions. Our analysis indicates that six out of the nine classical tertiary interactions are held in place mainly by H-bonds between the bases. In the remaining three cases other effects have to be considered. Tertiary base pairing interaction energies range from -8 to -38 kcal/mol in yeast tRNA(Phe) and are estimated to contribute roughly 25% of the overall tRNA base pairing interaction energy. Six analyzed posttranslational chemical modifications were shown to have minor effect on the geometry of the tertiary interactions. Modifications that introduce a positive charge strongly stabilize the corresponding tertiary interactions. Non-additive effects contribute to the stability of base triplets.

Base Pairing↗
PubMed1985

Nucleic acid and protein metabolism in undernutrition and protein deficiency.

This review discusses the metabolism of nucleic acids and proteins in various models of undernutrition in female rats and their neonatal and 21-day-old progeny. Based on the observations noted in our laboratories and those of other investigators, it is concluded that body and organ weights as well as various parameters of cellular growth (DNA, RNA, proteins, amino acids and total nucleotides) fail to increase normally in dietary-insulted animals. Protein and RNA synthesis demonstrate variable responses, leading to the speculation that modulation of mRNA metabolism and of protein synthesis occurs in dietary-restricted rats. These findings are also confirmed by the organ weight to DNA ratios. It is further noted that, despite the increases in protein and RNA synthesis in certain organs, protein and RNA register below-normal values, indicating that their degradation is much faster than their formation. This postulate is supported: by the enhanced activities of acid cathepsin (a protein-degrading enzyme) and of RNAse A (a RNA-degrading enzyme); by the elevated concentrations of circulating amino acids and total nucleotides; as well as by the accelerated excretion of nitrogenous compounds in the urine and feces of dietary-restricted animals. Modifications of RNA turnover are also evident in the tRNA and soluble RNA fractions of the liver of dietary-insulted rats. Studies on brain mRNA translatability have revealed: that food deprivation elicits a shorter species of pre-mRNA via a reduced polynucleotide elongation rate; that not all poly A+ RNA sequences present in control rats occur in dietary-restricted animals; and that the translatability of polymerase II is far lower in dietary-insulted rats. Other investigations on the translatability of liver, brain, kidney, spleen and thymus mRNA have demonstrated changes in mRNA via altered protein synthesis in various organs of dietary-restricted rats. Generation studies have shown that adaptation prevails in the first, second and third generation offspring of dietary-insulted rats, after which all parameters decline in fourth and fifth generation offspring. By reducing the litter size and exchanging the pups of control and dietary-restricted rats during the lactation period, partial restoration of the cellular growth of different organs is effected with the exception of the brain, in which damage is irreversible.

Animals↗
PubMed1997

Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.

Affinity Labels↗
PubMed2003

Identifying the methyltransferases for m(5)U747 and m(5)U1939 in 23S rRNA using MALDI mass spectrometry.

There are three sites of m(5)U modification in Escherichia coli stable RNAs: one at the invariant tRNA position U54 and two in 23S rRNA at the phylogenetically conserved positions U747 and U1939. Each of these sites is modified by its own methyltransferase, and the tRNA methyltransferase, TrmA, is well-characterised. Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA, and here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry. A purified recombinant version of YgcA retains its activity and specificity, and methylates U1939 in an RNA transcript in vitro. We were unable to generate a recombinant version of YbjF that retained in vitro activity, so the function of this enzyme was defined in vivo by engineering a ybjF knockout strain. Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification. With this report, the functions of all the E.coli m(5)U RNA methyltransferases are identified, and a more appropriate designation for YbjF would be RumB (RNA uridine methyltransferases B), in line with the recent nomenclature change for YgcA (now RumA).

Base Sequence↗
PubMed1990

The contacts of yeast tRNA(Ser) with seryl-tRNA synthetase studied by footprinting experiments.

Yeast tRNA(Ser) is a member of the class II tRNAs, whose characteristic is the presence of an extended variable loop. This additional structural feature raises questions about the recognition of these class II tRNAs by their cognate synthetase and the possibility of the involvement of the extra arm in the recognition process. A footprinting study of yeast tRNA(Ser) complexed with its cognate synthetase, yeast seryl-tRNA synthetase (an alpha 2 dimer), was undertaken. Chemical (ethylnitrosourea) and enzymatic (nucleases S1 and V1) probes were used in the experiments. A map of the contact points between the tRNA and the synthetase was established and results were analyzed with respect to a three-dimensional model of yeast tRNA(Ser). Regions in close vicinity with the synthetase are clustered on one face of tRNA. The extra arm, which is strongly protected from chemical modifications, appears as an essential part of the contact area. The anticodon triplet and a large part of the anticodon arm are, in contrast, still accessible to the probes when the complex is formed. These results are discussed in the context of the recognition of tRNAs in the aminoacylation reaction.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Metal ion stabilization of the U-turn of the A37 N6-dimethylallyl-modified anticodon stem-loop of Escherichia coli tRNAPhe.

Nucleoside base modifications can alter the structures, dynamics, and metal ion binding properties of transfer RNA molecules and are important for accurate aminoacylation and for maintaining translational fidelity and efficiency. The unmodified anticodon stem-loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg(2+) and dimethylallyl modification of A(37) N6 disrupt the loop conformation and increase the mobility of the loop and loop-proximal nucleotides. We have used NMR spectroscopy to investigate the binding and structural effects of multivalent cations on the unmodified and dimethylallyl-modified anticodon stem-loops from E. coli tRNA(Phe). The divalent cation binding sites were probed using Mn(2+) and Co(NH(3))(6)(3+). These ions bind along the major groove of the stem and associate with the anticodon loop on the major groove side in a nonspecific manner. Co(NH(3))(6)(3+) stabilizes the U-turn conformation of the loop in the dimethylallyl-modified molecule, and the chemical shift changes that accompany Co(NH(3))(6)(3+) binding are similar to those observed with the addition of Mg(2+). The base-phosphate and base-2'-OH hydrogen bonds that characterize the UNR U-turn motif lead to spectral signatures in the form of unusual (15)N and (1)H chemical shifts and reduced solvent exchange of the U(33) 2'-OH and N3H protons. The unmodified molecule also displays spectral features of the U-turn fold in the presence of Co(NH(3))(6)(3+), but the loop has additional conformations and is dynamic. The results indicate that charge neutralization by a polyvalent cation is sufficient to promote formation of the U-turn fold. However, base modification is necessary to destabilize competing alternative conformers even for a purine-rich loop sequence that is predicted to have strongly favorable base stacking energy.

Adenine↗
PubMedDate not supplied

[The first nucleotide of the codon located in the P site of the 80S ribosome is close to G1702 of the 18S rRNA as revealed by crosslinking to a pUUUGUU derivative containing a perfluorophenylazido group at the guanine].

Modification of the 18S rRNA with a pUUUGUU derivative carrying a perfluorophenylazido group at N7 of G was studied in the complex with the human 80S ribosome and Val-tRNA(Val), which directs modified GUU to the P site. Reverse transcription reported modification of invariant G1702 of the 18S rRNA. On evidence of the results and the earlier data on affinity modification of the human ribosome with tetra- or heptaribonucleotide derivatives carrying an alkylating group at the 3' end, the template was assumed to make a bend between the A- and P-site codons, which brings both codons closer to G1702 of the 18S rRNA.

Base Sequence↗