On the recognition of tRNA by its aminoacyl-tRNA ligase.
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Amino Acids↗
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Purification and properties of a neutral endodeoxyribonuclease from rat small intestinal mucosa.
An endodeoxyribonuclease has been purified to near homogeneity from rat small intestinal mucosa by a procedure involving Con A-Sepharose affinity chromatography. During the initial steps of purification, the presence of 5 mM CaCl2 was essential for stability of the enzyme activity. The enzyme has a molecular weight of 32 000 and an isoelectric point of 4.7. NaCl, sulfhydryl reagents, and iodoacetate strongly inhibited the reaction, but tRNA did not. The enzyme required divalent cations for activity and had a pH optimum of pH 6.2 with Co2+ and pH 7.7 with Mn2+. In both optimum conditions, the enzyme hydrolyzed native DNA more rapidly than denatured DNA, and the average chain lengths of limit digestion products of native and denatured DNA were 8 and 10, respectively, at pH 6.2 and 9 and 11, respectively, at pH 7.7. The enzyme activity to produce acid-soluble fractions from linear DNA substrate was similar in the two optimum conditions, but the activity to nick double-stranded, superhelical circular DNA substrate was significantly higher at pH 6.2 than at pH 7.7. The endonuclease formed single-strand breaks making 5'-phosphoryl and 3'-hydroxyl termini, and deoxythymidine was present at the 5' termini with a frequency of about 50% in both optimum conditions. Bovine pancreatic DNase I antibody and G-action inhibited the enzyme activity. Thus this endonuclease is classified as a DNase I.
Animals↗
Sequential processing of precursor tRNA molecules in Escherichia coli.
In a temperature-sensitive mutant of E. coli defective in tRNA biosynthesis, many tRNA precursors, including monomeric and multimeric forms, accumulate. Some of the multimeric precursors contain three or more tRNA sequences within a molecule. These large precursors were cleaved by cell extracts first into intermediate size pieces which were subsequently processed by RNase P. On the basis of heat stability of mutant cell extracts, the endonuclease responsible for the initial cleavage appears to be distinct from RNase P and is designated RNase O. One of the monomeric precursors was shown to be processed first by RNase P and the product subsequently cleaved further into a smaller molecule. The nuclease responsible for this second cleavage also appears to be distinct from RNase P and is designated RNase Q. The functions of these nucleases are sequential in the trimming process with respect to that of RNase P; RNase O works prior to RNase P and RNase Q after RNase P but in both cases, not vice versa.
Escherichia coli↗
Functional characterization of chimeric reverse transcriptases with polypeptide subunits of highly divergent HIV-1 group M and O strains.
Human immunodeficiency virus (HIV)-1 strains have been divided into three groups: main (M), outlier (O), and non-M non-O (N). Biochemical analyses of HIV-1 reverse transcriptase (RT) have been performed predominantly with enzymes derived from HIV-1 group M:subtype B laboratory strains. This study was designed to optimize the expression and to characterize the enzymatic properties of HIV-1 group O RTs as well as chimeric RTs composed of group M and O p66 and p51 subunits. The DNA-dependent DNA polymerase activity on a short heteropolymeric template-primer was similar with all enzymes, i.e. the HIV-1 group O and M and chimeric RTs. Our data revealed that the 51-kDa subunit in the chimeric heterodimer p66(M:B)/p51(O) confers increased heterodimer stability and partial resistance to non-nucleoside RT inhibitors. Chimeric RTs (p66(M:B)/p51(O) and p66(O)/p51(M:B)) were unable to initiate reverse transcription from tRNA(3)(Lys) using HIV-1 group O or group M:subtype B RNA templates. In contrast, HIV-1 group O and M RTs supported (-)-strand DNA synthesis from tRNA(3)(Lys) hybridized to any of their corresponding HIV-1 RNA templates. HIV-2 RT could not initiate reverse transcription on tRNA(3)(Lys)-primed HIV-1 genomic RNA. These findings suggest that the initiation event is conserved between HIV-1 groups, but not HIV types.
Base Sequence↗
A catalytic mechanism for D-Tyr-tRNATyr deacylase based on the crystal structure of Hemophilus influenzae HI0670.
D-Tyr-tRNA(Tyr) deacylase is an editing enzyme that removes d-tyrosine and other d-amino acids from charged tRNAs, thereby preventing incorrect incorporation of d-amino acids into proteins. A model for the catalytic mechanism of this enzyme is proposed based on the crystal structure of the enzyme from Haemophilus influenzae determined at a 1.64-A resolution. Structural comparison of this dimeric enzyme with the very similar structure of the enzyme from Escherichia coli together with sequence analyses indicate that the active site is located in the dimer interface within a depression that includes an invariant threonine residue, Thr-80. The active site contains an oxyanion hole formed by the main chain nitrogen atoms of Thr-80 and Phe-79 and the side chain amide group of the invariant Gln-78. The Michaelis complex between the enzyme and D-Tyr-tRNA was modeled assuming a nucleophilic attack on the carbonyl carbon of D-Tyr by the Thr-80 O(gamma) atom and a role for the oxyanion hole in stabilizing the negatively charged tetrahedral transition states. The model is consistent with all of the available data on substrate specificity. Based on this model, we propose a substrate-assisted acylation/deacylation-catalytic mechanism in which the amino group of the D-Tyr is deprotonated and serves as the general base.
Amino Acid Sequence↗
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↗
Isoleucyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus. I. Properties of the enzyme.
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Adenosine Monophosphate↗
The glucocorticoid receptor protein binds to transfer RNA.
The glucocorticoid receptor from mouse AtT-20 cells exists in three forms: the untransformed receptor (9.1S; Mr of 319,000), a large oligomeric molecule that does not bind to DNA; the transformed receptor (4S; Mr of 96,000), which is formed by dissociation of untransformed receptor after steroid binding and which binds to DNA to modulate gene expression; and an intermediate size receptor (6S; Mr of 132,000), which also binds to DNA and contains a bound small RNA molecule. This RNA species has now been purified and identified as transfer RNA (tRNA). The three tRNA's for the basic amino acids accounted for about 78% of the total amino acid-accepting activity [arginine (52%), lysine (17%), and histidine (9%)], while the remaining 22% was represented by six other tRNA species. This tRNA-binding activity of the glucocorticoid receptor may reflect post-transcriptional mechanisms of regulating gene expression, such as alterations in the translational efficiency of or the modulation of the stability of hormone-induced proteins.
Animals↗
Temporal appearance of bacteriophage T4-modified valyl tRNA synthetase in Escherichia coli.
Bacteriophage T4-induced modification of Escherichia coli vlayl-tRNA synthetase (EC 6.1.1.9) requires: synthesis of a phage-gene specified tau factor, addition of the factor to host valyl-tRNA synthetase to produce a urea-stable enzyme, and interaction of the modified enzyme with tRNA to produce a more rapidly sedimenting valyl-tRNA synthetase activity on sucrose density gradients. This report demonstrates that the coincident, chloramphenicol-sensitive appearance of urea-stable and rapidly sedimenting valyl-tRNA synthetase activity are immediate early phage functions. It implies that once the tau factor is synthesized, further interactions are stoichiometric rather than catalytic. The potential for valyl-tRNA synthetase modification accumylates when E. coli is infected with T4 PHAGE IN THE PRESENCE OF CHLORAMPHINICOL AND IS EXPRESSED DURING THE RESUMPTION OF PROTEIN SYNTHESIS WHEREAS FURTHER RNA synthesis is inhibited by rifampicin. The modification phenomenon occurs similarly in several strains of E. coli and represents a novel virus-host interaction.
Amino Acyl-tRNA Synthetases↗
The so-called tRNAGlu1 of Escherichia coli is a stable denatured conformer of the major isoacceptor tRNAGlu2.
A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This tRNAGluUUC (or tRNAGluSUC when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli.
Carbon Radioisotopes↗
Differential stability of mitochondrial mRNA in HeLa cells.
The physiological significance and metabolism of oligoadenylated and polyadenylated human mitochondrial mRNAs are not known to date. After study of eight mitochondrial transcripts (ND1, ND2, ND3, ND5, CO1, CO2, ATP6/8 and Cyt. b) we found a direct correlation between the half-lives of mitochondrial mRNAs and their steady-state levels. Investigation of the mt-mRNA decay after thiamphenicol treatment indicated that three transcripts (ND2, ND3 and Cyt. b) are significantly stabilized after inhibition of mitochondrial translation. Careful analysis one of them, ND3, showed that inaccurate processing of the H-strand RNA precursor may occasionally occur between the ND3 and tRNA(Arg) locus leading to synthesis of ND3 mRNAs lacking the STOP codon. However, analysis of the oligo(A) fraction observed in case of the ND3 indicates that partially polyadenylated mRNAs are linked rather to the transcription process than to the translation-dependent deadenylation. Analysis of ND3 mRNA turnover in cells with siRNA-mediated knock-down of the mitochondrial poly(A) polymerase shows that strongly decreased polyadenylation does not markedly affect the decay of this transcript. We present a model where oligoadenylated mitochondrial transcripts are precursors of molecules containing full length poly(A) tails.
Blotting, Northern↗
The structure of 3'-O-anthraniloyladenosine, an analogue of the 3'-end of aminoacyl-tRNA.
3'-O-Anthraniloyladenosine, an analogue of the 3'- terminal aminoacyladenosine residue in aminoacyl-tRNAs, was prepared by chemical synthesis, and its crystal structure was determined. The sugar pucker of 3'-O-anthraniloyladenosine is 2'-endo resulting in a 3'-axial position of the anthraniloyl residue. The nucleoside is insynconformation, which is stabilized by alternating stacking of adenine and benzoyl residues of the neighboring molecules in the crystal lattice. The conformation of the 5'-hydroxymethylene in 3'-O- anthraniloyladenosine is gauche-gauche. There are two intramolecular and two intermolecular hydrogen bonds and several H-bridges with surrounding water molecules. The predominant structure of 3'-O-anthraniloyladenosine in solution, as determined by NMR spectroscopy, is 2'-endo,gauche-gauche and anti for the sugar ring pucker, the torsion angle around the C4'-C5'bond and the torsion angle around the C1'-N9 bond, respectively. The 2'-endo conformation of the ribose in 2'(3')-O-aminoacyladenosine, which places the adenine and aminoacyl residues in equatorial and axial positions, respectively, could serve as a structural element that is recognized by enzymes that interact with aminoacyl-tRNA or by ribosomes to differentiate between aminoacylated and non-aminoacylated tRNA.
Acylation↗
Histidine Supplementation Stabilizes Hearing and Vision and Improves Growth in HARS1-Related Autosomal Recessive Disorder Associated With Usher-Like Symptoms.
Autosomal recessive HARS1-related disorder (originally described as Usher syndrome type 3B) caused by a homozygous Y454S variant in the histidyl-tRNA synthetase gene (HARS1) is characterized by progressive sensorineural hearing and vision loss and respiratory deterioration with risk for sudden death following febrile illnesses. In-vitro studies have previously shown that histidine can rescue a humanized yeast model for pathogenic HARS alleles. Fourteen children homozygous for HARS Y454S were treated with supplemental oral histidine (50 mg/kg BID) and monitored with bloodwork and physical, visual, and audiometry assessments during a 3-year clinical trial, then followed for more than 4 years on histidine in the post-trial period. Patient fibroblasts were assessed for response to histidine. Hearing and vision remained stable, and growth improved significantly. Children remained healthy, with no severe deteriorations despite exposure to bacterial and viral infections, including COVID-19. Gains in growth were maintained in the post-trial period on varying levels of histidine supplementation. Daily oral histidine supplementation in children with autosomal recessive HARS1-related disorder can ameliorate or slow the progression of disease and is safe, inexpensive, and well tolerated. This study adds to the growing list of autosomal recessive ARSopathies (aminoacyl-tRNA synthetase disorders) that are amenable to amino acid supplementation.
Humans↗
Differential binding of a S. cerevisiae RNA polymerase III transcription factor to two promoter segments of a tRNA gene.
A Saccharomyces cerevisiae protein fraction which binds specifically to the internal promoter regions of genes that are transcribed by RNA polymerase III is shown to function as a transcription factor. We postulate that the stable DNA binding of the factor confers stability on polymerase III transcription complexes. Analysis of the binding by DNase 'foot-printing' distinguishes three segments of the S. cerevisiae tRNALeu3 gene: a region surrounding the so-called A block of the internal promoter, a region surrounding the B block and an intermediate segment. Binding to the A and B block regions is connected, but the B block region exerts a dominant effect.
Base Sequence↗
Binding of tRNA in different functional states to Escherichia coli ribosomes as measured by velocity sedimentation.
The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex. fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310-5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe. EF-Tu x guanosine 5'-[beta,gamma-methylene]triphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.
Binding Sites↗
Calorimetric investigations on thermal stability of tRNAIle (yeast) and tRNASer (yeast).
Variation with temperature of the partial heat capacities of tRNAIle (yeast) and tRNASer (yeast) has been determined in two buffers at various salt conditions by scanning microcalorimetry. The overall molar transition enthalpy, deltaHt = 320 +/- 20 kcal mol-1 (1339 +/- 84 kJ mol-1) is identical for the two tRNA species within the limits of experimental error. deltaHt does not show any dependence on the nature of the buffer, nor does it vary on addition of 1 mM MgCl2 or 150 mM NaCl. Thermal unfolding of the native structure to the random coil cannot adequately be described by a two-state, concerted transition under the experimental conditions applied in this study, but exhibits a multistep mechanism characterized by sequential unfolding of separable cooperative domains.
Calorimetry↗
Temperature-sensitive mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 genes result in defects in mRNA turnover.
In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37 degrees. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p.
Amino Acid Sequence↗
Modified 5'-nucleotides resistant to 5'-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl) uridine 5'-phosphate and N2, N2-dimethylguanosine 5'-phosphate from snake venom hydrolysates of transfer RNA.
A procedure for the quantitative measurement of the O2'-methylnucleoside constitutents of RNA has recently been developed in this laboratory (Gray, M.W. Can. J. Biochem. 53, 735-746 (1975)). This assay method is based on the resistance of O2'-methylnucleoside 5'-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5'-nucleotidase (EC 3.1.3.5). In the present investigation, two base-modified 5'-nucleotides, each displaying an unusual resistance to 5'-nucleotidase, have been identified. These compounds have been characterized by a variety of techniques as N2, N2-dimethylguanosine 5'-phosphate (pm2/2G) and 3-(3-amino-3-carboxypropyl)uridine 5'-phosphate (p4abu3U). Because of their resistance to 5'-nucleotidase, pm2/2G and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA. Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide. The proportion (mean +/- SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 +/- 0.03 (n=5) and 0.14 +/- 0.02 (n=4) mol%, respectively. The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data. The variable recovery of pm2/2G from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistent to 5'-nucleotidase. The complete absence of pm2/2G in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2, N2-dimethylguanosine in this RNA. These observations demonstrate that resistance to 5'-nucleotidase is a necessary but not sufficient criterion for concluding that a 5'-nucleotide is O2'-methylated. When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U. It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A.G. & Enger, M.D. Biochim. Biophys. Acta 349, 61-77 (1974)), may not be present in wheat embryo ribosomal RNA.
Guanine Nucleotides↗