2'-Versus 3'-OH specificity in tRNA aminoacylation. Further support for the "secondary cognition" proposal.

Purified Escherichia coli tRNAAla and tRNALys were each converted to modified species terminating in 2'- and 3'-deoxyadenosine. The modified species were tested as substrates for activation by their cognate aminoacyl-tRNA synthetases and for misacylation with phenylalanine by yeast phenylalanyl-tRNA synthetase. E. coli alanyl- and lysyl-tRNA synthetases normally aminoacylate their cognate tRNA's exclusively on the 3'-OH group, while yeast phenylalanyl-tRNA synthetase utilizes only the 2' position on its own tRNA. Therefore, the finding that the phenylalanyl-tRNA synthetase activated only those modified tRNAAla and tRNALys species terminating in 3'-deoxyadenosine indicated that the position of aminoacylation in this case was specified entirely by the enzyme, an observation relevant to the more general problem of the reason(s) for using a particular site for aminoacylation and maintaining positional specificity during evolution. Initial velocity studies were carried out using E. coli tRNAAla and both alanyl- and phenylalanyl-tRNA synthetases. As noted in other cases, activation of the modified and unmodified tRNA's had essentially the same associated Km values, but in each case the Vmax determined for the modified tRNA was smaller.     

Reaction of nucleic acid bases with alpha-acetylenic esters. Part IV. Preparation of an alkylating derivative of tRNA(Phe) by conformation-specific chemical modification

The reaction of yeast tRNA(Phe) with methyl chlorotetrolate, ClCH2-C identical to C-COOCH3, was studied. This reagent converts adenine and cytosine rings into derivatives in which an additional heterocycle bearing the alkylating chloromethyl group is fused to the original base; these derivatives can exist in two isomeric forms. Modified nucleosides of this type can be easily identified by reverse-phase HPLC. It was found that under native conditions, the modification of tRNA involves the anticodon loop and the 3'-end. The isomers of adenine derivatives formed in the anticodon loop were different from those formed in the 3'-end. It is suggested that the isomeric structure of the derivatives is related to the fine conformational differences between these two regions of tRNA(Phe). Methyl chlorotetrolate could thus be used as a conformational probe of single-stranded nucleic acids. Preliminary assays showed that modified tRNA(Phe) binds irreversibly to yeast phenylalanyl-tRNA synthetase.     

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by E. coli tRNAphe containing photoreactive group.

The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C.6.1.1.20) from E.coli MRE-600 with tRNAphe containing photoreative p-N3-C6H4-NHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAphe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNAphe with the enzyme, however it results in sevenfold increase of Km in the enzymatic aminoacylation of tRNAphe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [14C]-Phe-azido-tRNAphe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 xx 10(-3) M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exhibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [14C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA enzyme interaction.     

Phenylalanyl-tRNA synthetase from E. coli MRE-600. Effect of chemical modification of lysine residues on the enzyme interaction with substrates

The effect of modification of Phe-RSase from E. coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated. It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-[32P]-pyrophosphate exchange reaction. The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction. tRNAphe strongly protects the enzyme against inactivation. ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect. L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP. The dissociation constant of the Phe-RSase[14C]-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction. There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested. Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex. Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction. It is assumed that Phe-RSase from E. coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center.     

A strong ethidium binding site in the acceptor stem of most or all transfer RNAs.

E. coli unfractionated tRNA and tRNA phe both contain a single strong ethidium binding site. Singlet-singlet energy transfer has been used to measure the distance between this site and dansyl hydrazine covalently attached to the 3' end of the tRNAs. The distance obtained is between 33 and 40 A for both samples. This is completely consistent with results from earlier NMR studies which placed the single, strong ethidium binding site of yeast tRNAphe between base pairs 6 and 7 on the aminoacyl stem. From the known tertiary structure of tRNAphe it is possible to rationalize the unusual affinity of this site and its likely existence in all tRNAs.     

Modification of ribonucleic acid by chemical carcinogens. VI. Effect of N-2-acetylaminofluorene modification of guanosine on the codon function of adjacent nucleosides in oligonucleotides

Oligonucleotides containing a guanosine residue on the 5′ or the 3′ side of tri- and tetranucleotides were prepared. The guanosine residue was modified with the chemical carcinogen N-2-acetylaminofluorene and the control and modified oligonucleotides were tested for their ability to stimulate ¹⁴C-labeled amino-acyl-tRNA binding to ribosomes. The effects of the modification are twofold. The first is that if the guanosine residue to which the drug is eovalently bound is part of a codon the oligonucleotide is completely inactive in the ribosomal binding assay. The second is that if an adenosine residue is adjacent to either the 5′ or 3′ side of the modified guanosine, as in (Ap)₃G or G(pA)₃, there is partial inhibition of ¹⁴C-labeled lysyl-tRNA binding to ribosomes. This inhibitory effect extends only to the function of the immediately adjacent adenosine since the chemical modification of guanosine residues in (Ap)₄G or G(pA)₄ did not impair their ability to code for lysine. In contrast to these findings if there is a uridine residue adjacent to the modified guanosine, as in (Up)₃G or G(pU)₃ there is no effect on ¹⁴C-labeled phenylalanyl-tRNA binding to ribosomes. Proton magnetic resonance spectra of UpG, GpU and the corresponding dinners in which the guanosine residue was modified with the drug failed to indicate a stacking interaction between the fluorene moiety and the adjacent uridine residue. This is in contrast to previous studies demonstrating a strong stacking interaction between fluorene and adjacent adenosine residues. Taken together these results indicate that acetylaminofluorene modification of guanosine next to an adenosine residue in oligonucleotide inhibits its ribosomal binding capacity. The stacking interaction with adjacent adenosine, and not with adjacent uridine residues, in oligonucleotides probably accounts for the effects observed in the ribosomal binding assay. These data are consistent with our previously described “base displacement” model.     

An NMR study of the exchange rates for protons involved in the secondary and tertiary structure of yeast tRNA Phe.

Solvent exchange rates of all the protons of yeast tRNAphe resonating in the lowfield NMR region (-11 to-15 ppm from DSS) have been measured by saturation-recovery long-pulse Fourier transform NMR. All these protons in yeast tRNAphe are in the fast exchange limit with H2O relative to their intrinsic longitudinal relaxation processes. Most rates show very little temperature dependence; however, tertiary base pair protons are preferentially destabilized in the absence of Mg++ at higher temperatures. The measured exchange rates are between 2 and 125 sec-1 for a temperature range from 10 degrees C to 45 degrees C and MgCl2 concentrations between 0 and 15 mM.     

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.     

360 MHz PMR studies on the involvement of the Y-nucleoside in the conformation of 2''-OMeGpApApYpAppsi from torula yeast tRNAphe.

360 MHz measurements of chemical shifts, 3J1'-2', and T1 as a function of temperature for various protons of the hexanucleotide 2'-OMeGpApApYpAppsi from torula yeast tRNAphe have revealed a unique involvement of the Yt base in the structure and conformation of this oligonucleotide. Whereas the adenosine residues in the anticodon triplet are relatively stable to temperature increase, the Yt readily undergoes destacking and a change in ribose conformation. The destacking most likely involves a torsional displacement of the Yt base occasioned by a rotation of the phosphate-ribose backbone. The possible relevance of this unusual behavior to the influence of the Yt residue in tRNA function in protein biosynthesis is discussed.     

Specific interaction of anticodon loop residues with yeast phenylalanyl-tRNA synthetase

Thirteen different yeast tRNAPhe variants with single nucleotide changes in positions 34-37 in the anticodon region were prepared by an enzymatic procedure described previously. Aminoacylation kinetics using purified yeast phenylalanyl-tRNA synthetase revealed that the level of aminoacylation was very different for different sequences inserted. The low level of aminoacylation was the result of a steady state between a slow forward reaction rate and spontaneous deacylation of the product. Aminoacylation kinetics performed at higher synthetase concentrations revealed that substitution at position 34 in tRNAPhe decreased the Km nearly 10-fold but only had a small effect on Vmax. Similar substitutions at positions 35, 36, and 37 had a lesser effect. These data suggest a sequence-specific contact between the anticodon of yeast tRNAPhe and the cognate synthetase.     

Accessible and inaccessible bases in yeast phenylalanine transfer RNA as studied by chemical modification.

The selective modification of cytidine, uridine, guanosine and dihydrouridine residues in ³²P-labelled yeast phenylalanine transfer RNA has been studied by the use of specific reagents.The selective modification of cytidine residues with the reagent methoxyamine is described. Of the six cytidines in the single-stranded regions of the cloverleaf formula, only two are completely reactive, C74 and C75 at the 3′-terminus. Cm32 in the anticodon loop is reactive to only a small extent.The selective modifications of uridine and guanosine residues with 1-cyclohexyl 3-[2-morpholino(4)-ethyl] carbodiimide methotosylate, is described. The reagent is also shown to be reactive with dihydrouridine. In the single-stranded regions of the secondary structure of yeast phenylalanine transfer RNA there are 16 base residues which this reagent could be specific for. However, only G20, Gm34 and U47 are extensively modified, whilst U33 and D16 are partially modified. G18 is modified to a very small extent.The results obtained in this study are also in good agreement with previous chemical modification studied by other workers, carried out on unlabelled yeast phenylalanine transfer RNA using different reagents to the ones described here.The pattern of chemical modification is compared with the three-dimensional structure obtained by an X-ray crystallographic analysis of the same tRNA species. The correlation between exposed regions of the model and the regions of chemical reactivity are everywhere consistent.     

Co-operative effects in affinity labeling reveal the interaction of tRNA-recognition centers of phenylalanyl-tRNA synthetase

A mathematical treatment of affinity labeling of the enzymes is presented. The model considered involves a dimeric enzyme with identical ligand binding sites. Equations are derived which describe the kinetics of modification; mutual influence of ligand molecules on association, on the rate of covalent attachment and the possibility of the existence of different sites of modification are taken into account. Experimental data on affinity labeling of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) of Escherichia coli MRE-600 with N-bromoacetyl-[14C]phenylalanyl-tRNA are treated in terms of the model suggested. The affinity (association constant value) of the tRNAPhe analog molecule towards the enzyme is only slightly affected by another molecule, whereas the reaction rate constant of covalent attachment decreases significantly. The latter is assumed to be due to acceptor site change in the complex containing two molecules of the tRNAPhe analog.     

QM/MM and free-energy simulations of deacylation reaction catalysed by sedolisin,a serine-carboxyl peptidase

Quantum mechanical/molecular mechanical free-energy simulations were performed to understand the deacylation reaction catalysed by sedolisin (a serine-carboxyl peptidase) and to elucidate the catalytic mechanism and the role of the active-site residues during the process. The results given here demonstrate that Asp170 may act as a general acid/base catalyst for the deacylation reaction. It is also shown that the electrostatic oxyanion hole interactions involving Asp170 may be less effective in transition state stabilisation for the deacylation step in the sedolisin-catalysed reaction compared to the general acid/base mechanism. The proton transfer processes during the enzyme-catalysed process were examined, and their role in the catalysis was discussed.     

A cluster of six tRNA genes in Drosophila mitochondrial DNA that includes a gene for an unusual tRNAserAGY.

Genes for URF3, tRNAala, tRNAarg, tRNAasn, tRNAserAGY, tRNAglu, tRNAphe, and the carboxyl terminal segment of the URF5 gene have been identified within a sequenced segment of the mtDNA molecule of Drosophila yakuba. The genes occur in the order given. The URF5 and tRNAphe genes are transcribed in the same direction as replication while the URF3 and remaining five tRNA genes are transcribed in the opposite direction. Considerable differences exist in the relative arrangement of these genes in D. yakuba and mammalian mtDNA molecules. In the tRNAserAGY gene an eleven nucleotide loop, within which secondary structure formation seems unlikely, replaces the dihydrouridine arm, and both the variable loop (six nucleotides) and the T phi C loop (nine nucleotides) are larger than in any other D. yakuba tRNA gene. As available evidence is consistent with AGA codons specifying serine rather than arginine in the Drosophila mitochondrial genetic code, the possibility is considered that the 5'GCU anticodon of the D. yakuba tRNAserAGY gene can recognize AGA as well as AGY codons.     

T4 RNA ligase mediated preparation of novel "chemically misacylated" tRNAPheS

T4 RNA ligase was employed for the condensation of Escherichia coli tRNAPhe missing cytidine-75 and adenosine-76 (tRNAPhe-COH; the acceptor "oligomer") with each of several chemically acylated derivatives of pCpA (the donor "oligomer"). The resulting "chemically misacylated " tRNAPheS were obtained in 20-65% yields following chromatographic workup on DEAE-cellulose and benzoylated DEAE-cellulose. Characterization of the chemically misacylated tRNAs was accomplished by (i) enzymatic reaminoacylation of chemically misacylated tRNAPhe with phenylalanine by E. coli phenylalanyl-tRNA synthetase following chemical deacylation of the "incorrect" amino acid, (ii) comparison of the hydrolytic effects of Cu2+ solutions on chemically and enzymatically prepared samples of N-acetyl-L-phenylalanyl- tRNAPheS , and (iii) measurement of the chromatographic behavior of the tRNA species derived from chemical misacylation .     

Primary structure of yeast mitochondrial DNA-coded phenylalanine-tRNA.

Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase.     

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [¹⁴C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the ¹⁴C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.     

1H NMR studies of transfer RNA III: the observed and the computed spectra of the hydrogen-bonded NH resonances of baker''s yeast transfer-RNA Phe.

The hydrogen-bonded NH resonances of Baker's yeast tRNAphe in H2O solution with Mg++ have been measured by a 360 MHz spectrometer at 23 degrees C. Totally, fifteen peaks and one shoulder can be resolved which represent 25 +/- 1 protons. Based on the refined atomic coordinates of the tRNAphe in the orthorhombic crystal, on the recent advances in the distance dependence of the ring-current magnetic field effects and on the adopted values for the isolated hydrogen-bonded NH resonances, a computed spectrum consisting of 23 protons was constructed. A quantitative comparison by computer was made between the computed spectrum and the spectrum simulated from the observed spectrum. These two spectra are closely similar but not identical. We suggest that the conformation of yeast tRNAphe in aqueous solution is closely similar but not identical to that found in the crystal, especially in the T psi C region and D region. Also the NH resonances in 3-4 proposed hydrogen bonds (most likely for tertiary structure) may exchange very rapidly in aqueous solution.