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.     

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs and aminoacyl-tRNA synthetases. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA(Pro) with cysteine, but does not aminoacylate archaeal tRNA(Cys) with cysteine; (iv) identification and characterization of the AUA-decoding minor tRNA(Ile) in archaea; and (v) evidence that the archaeal minor tRNA(Ile) contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.     

Transfer RNA aminoacylation: identification of a critical ribose 2'-hydroxyl-base interaction.

To understand the relationship between tRNA architecture and specific aminoacylation by aminoacyl-tRNA synthetases, we performed kinetic assays of Escherichia coli tRNA(Pro) molecules containing single deoxynucleotide substitutions. We identified an important 2'-hydroxyl group at position U8 (of 22 positions probed). Chemical modification studies showed that this 2'-hydroxyl interacts with either the N1 or the exocyclic amine of G46 in a hydrogen bonding interaction that contributes 1.8 kcal/mol to the free energy of activation for aminoacylation. Molecular modeling of tRNA(Pro) supports the existence of this interaction. This is the first study to identify a specific ribose 2'-hydroxyl-base interaction in the core region of a tRNA molecule that makes a thermodynamically significant contribution to aminoacylation.     

Commonality and diversity in tRNA substrate recognition in t6A biogenesis by eukaryotic KEOPSs

N ⁶-Threonylcarbamoyladenosine (t⁶A) is a universal and pivotal tRNA modification. KEOPS in eukaryotes participates in its biogenesis, whose mutations are connected with Galloway-Mowat syndrome. However, the tRNA substrate selection mechanism by KEOPS and t⁶A modification function in mammalian cells remain unclear. Here, we confirmed that all ANN-decoding human cytoplasmic tRNAs harbor a t⁶A moiety. Using t⁶A modification systems from various eukaryotes, we proposed the possible coevolution of position 33 of initiator tRNA^Met and modification enzymes. The role of the universal CCA end in t⁶A biogenesis varied among species. However, all KEOPSs critically depended on C32 and two base pairs in the D-stem. Knockdown of the catalytic subunit OSGEP in HEK293T cells had no effect on the steady-state abundance of cytoplasmic tRNAs but selectively inhibited tRNA^Ile aminoacylation. Combined with in vitro aminoacylation assays, we revealed that t⁶A functions as a tRNA^Ile isoacceptor-specific positive determinant for human cytoplasmic isoleucyl-tRNA synthetase (IARS1). t⁶A deficiency had divergent effects on decoding efficiency at ANN codons and promoted +1 frameshifting. Altogether, our results shed light on the tRNA recognition mechanism, revealing both commonality and diversity in substrate recognition by eukaryotic KEOPSs, and elucidated the critical role of t⁶A in tRNA^Ile aminoacylation and codon decoding in human cells.     

Regulation of RNA function by aminoacylation and editing?

The chemical modification of nucleic acids is a ubiquitous phenomenon. Aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a reaction essentially devoted to protein synthesis but it is used also as an emergency mechanism to recycle stalled ribosomes, and it is required for genome replication in some RNA viruses. In several aminoacyl-tRNA synthetases a correction mechanism known as editing is present to prevent aminoacylation errors. Genome data reveal a growing number of open reading frames encoding ARS-like proteins. This strongly suggests the existence of a widespread and nonconventional machinery for aminoacylation and editing. Here we review the different biological functions of aminoacylation and editing; also we propose an evolutionary scenario for the origin of these two reactions, and hypothesize an extant role for RNA charging and editing outside the genetic code.     

Studies on the effects ofin vitro methyiation on aminoacylation of transfer RNA

In vitro methyiation ofEscherichia coli transfer ribonucleic acid by cell free extracts ofMycobacterium smegmatis leads exclusively to the formation of 1-methyl adenine [Vani, B. R., Ramakrishnan, T., Taya, Y., Noguchi, S., Yamaiuzumi, Z. and Nishimura, S. (1978)J. Bact., 137, 1085]. We have studied the effect of this modification on aminoacylationof Escherichia coli tRNA by mycobacterial enzymes. Aminoacylation with total algal protein hydrolysate as well as several individual aminoacids like methionine, valine, tyrosine, aspartic acid and lysine were monitored. In all the cases methyiation had a positive effect on the extent of aminoacylation by mycobacterial enzymes. Decreased aminoacylationin vitro was observed when hypomethylated transfer RNA from ethionine treated cells was used as the substrate for aminoacylation     

Tyrosyl-tRNA-synthetase from bovine liver. Functional role of histidine residues]

A specific chemical modification of histidyl residues in tyrosyl-tRNA synthetase by diethyl pyrocarbonate was performed. It is shown that five of sixteen histidyl residues can react with diethyl pyrocarbonate in the native conditions. Modification of two histidyl residues per dimer results in the inactivation of tyrosyl-tRNA synthetase in both steps of the tRNATyr aminoacylation. All substrates protect tyrosyl-tRNA synthetase against inactivation with diethyl pyrocarbonate, the most effective protector being combination of ATP and tyrosine. Histidyl residues of tyrosyl-tRNA synthetase are suggested to be involved in the catalytic mechanism of aminoacylation of tRNATyr.     

The three cysteine residues of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae are not essential for its activity

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63,000) each of these containing three cysteines (residues 255, 512 and 519 in the amino acid sequence). Thiol-specific probes were used to label these cysteines and study the resulting effect of the modification on the kinetic parameters of both the ATP/PPi exchange and tRNA aminoacylation reactions. Using the classical techniques of protein chemistry it was shown that none of the three cysteines was labelled with iodoacetic acid, whilst N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) reacted with Cys512 and Cys255, respectively. Only the latter modification was accompanied by a decrease in the rates of both enzyme activities whilst the Km values for the various substrates remained unaffected. Site-directed mutagenesis was also used to replace each of the three cysteines by other residues, either individually or simultaneously. For these experiments the enzyme was expressed in Escherichia coli using an expression vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. The resulting substitution in the amino-terminal part of the expressed enzyme had no effect on the kinetic parameters, compared to those of the enzyme purified from S. cerevisiae. Taking into account the consequences of such substitutions, as well as those of chemical modifications on the two reactions catalysed by the enzyme. ATP/PPi exchange and tRNA aminoacylation, it could be concluded that none of these three cysteines plays any essential role in either substrate binding or catalysis.     

Effects of modification of 4-thiouridine in E. coli tRNAfMet on methylation reaction with a thermostable Gm-methylase

Gm-methylase isolated from an extreme thermophile, Thermus thermophilus HB 27 methylates the 2'-OH of the ribose of G18 in the consensus GG sequence in the D loop, by recognizing the D loop-and-stem structure as a minimal substrate. Modification of s4U8 of E. coli tRNAfMet with S-benzylthioisothiourea resulted in a considerable decrease in methylation activity of Gm-methylase. The effect was cancelled by reduction with beta-mercaptoethanol. However, aminoacylation activity and methylation activity of m1A-methylase were scarcely influenced by the modification. These results suggest the involvement of s4U8 residue of tRNA in the recognition of Gm-methylase.     

Inhibition of transfer messenger RNA aminoacylation and trans-translation by aminoglycoside antibiotics

Transfer messenger RNA (tmRNA) directs the modification of proteins of which the biosynthesis has stalled or has been interrupted. Here, we report that aminoglycosides can interfere with this quality control system in bacteria, termed trans-translation. Neomycin B is the strongest inhibitor of tmRNA aminoacylation with alanine (K(i) value of approximately 35 micro m), an essential step during trans-translation. The binding sites of neomycin B do not overlap with the identity determinants for alanylation, but the aminoglycoside perturbs the conformation of the acceptor stem that contains the aminoacylation signals. Aminoglycosides reduce the conformational freedom of the transfer RNA-like domain of tmRNA. Additional contacts between aminoglycosides and tmRNA are within the tag reading frame, probably also disturbing reprogramming of the stalled ribosomes prior protein tagging. Aminoglycosides impair tmRNA aminoacylation in the presence of all of the transfer RNAs from Escherichia coli, small protein B, and elongation factor Tu, but when both proteins are present, the inhibition constant is 1 order of magnitude higher. SmpB and elongation factor Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation.     

Fluoresceinylthiocarbamyl-tRNATyr: a useful derivative of tRNATyr (E.coli) for physicochemical studies.

Fluoresceinylthiocarbamyl-tRNATyr (FTC-tRNATyr) is prepared from tRNATyr and fluoresceinisothiogyanate (FITC) under mildly alkaline conditions. Labelling occurs specificly at the base Q of tRNATyr. The modified tRNA is fully active in the aminoacylation assay; when aminoacylated it is recognized by the elongation factor Tu (EF-Tu). Codon-anticodon interaction, however, is severely affected by the modification.     

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.     

In Vivo Aminoacylation of Transfer Ribonucleic Acid in Bacillus subtilis and Evidence for Differential Utilization of Lysine-Isoaccepting Transfer Ribonucleic Acid Species

The presence or absence of certain amino acids has different effects on the ability of Bacillus subtilis to sporulate, and the intracellular pool size of amino acids has been reported to vary during sporulation. The idea that these variations might exert a regulatory effect through aminoacylation of transfer ribonucleic acid (tRNA) was investigated by studying the levels of aminoacylation in vivo in the logarithmic or stationary phase of growth. Both the periodate oxidation method and the amino acid analyzer were used to evaluate in vivo aminoacylation. The results indicated that in general the level of aminoacylation of tRNA's remained constant through stage III of sporulation, although there were detectable variations for specific amino acid groups. Our studies also showed that periodate oxidation damaged certain tRNA's; therefore, the results obtained by such a method should be interpreted with caution. Because the damage can affect certain isoaccepting species specifically, the periodate oxidation method cannot be used to establish which isoaccepting species are acylated in vivo. We also investigated the possibility of preferential use of particular tRNA species by polyribosomes. These results demonstrated a preferential use of lysyl-tRNA's at different growth stages. Control mechanisms operating during the early stages of sporulation, therefore, do not affect the overall level of aminoacylation. However, there is an effect on the levels of aminoacylation of specific amino acids and on which isoaccepting species are utilized by the polyribosome system.     

Stimulation of valyl- and isoleucyl-tRNA synthetase reactions by polyamines

The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg²⁺, polyamines significantly stimulate the reaction. Of the cations tested, only Mn²⁺, Co²⁺ and Ca²⁺ can partially substitute for Mg²⁺ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg²⁺ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg²⁺ is also prevntedvby spemine.     

The role of queuine in the aminoacylation of mammalian aspartate transfer RNAs.

Can a queuine-specific tRNA function normally without replacement of G by Q in its structure? To answer this, kinetics of aspartate queuine-containing tRNA (Q-tRNA) is compared with its queuine-deficient counterpart (G-tRNA). The results indicate that Asp Q-tRNA is a more effective substrate than the Asp G-tRNA. The Asp Q-tRNA exhibits a higher reaction velocity (Vmax greater than 30%) and a higher reaction rate (Km less than 55%) than its counterpart. The Asp tRNAs derived from human tumor lines and grown in athymic mice contain a full complement of queuine. This tumor tRNA exhibits aminoacylation kinetics similar to a normal liver tRNA. Reasons for observing the lack of a G-to-Q modification in cancer tRNAs by others are hypothesized. Two purified Asp isoacceptors from liver are compared for the aminoacylation reaction; small differences are noted in the Vmax, but none in the Km values.     

Chemical modification of lysine residues in tyrosyl-tRNA-synthetase from cattle liver using pyridoxal-5'-phosphate]

Chemical modification of lysine residues of eukaryotic tyrosyl-tRNA synthetase was studied. It was shown that only four out of 22 lysine residues per enzyme dimer could be modified with pyridoxal-5'-phosphate. This modification led to the inactivation of tRNATyr aminoacylation by more than 90% but did not practically affect the rate of ATP-[32P]pyrophosphate exchange. Low molecular weight substrates (ATP, ATP-tyrosine) weakly protected the enzyme from inactivation, whereas tRNATyr afforded a much more effective protection. It was supposed that lysine residues of tyrosyl-tRNA synthetase can be involved in the interaction with tRNATyr.     

An important 2'-OH group for an RNA-protein interaction

We have investigated the role of 2′-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNA^Cys and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2′-OH by 2′-deoxy or 2′-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2′-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2′-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2′-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2′-OH group instead provides a monitor of the steric environment during the RNA–synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2′-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.     

Interactions of aminoacyl-tRNA synthetases in high-molecular-weight multienzyme complexes from rat liver

The functional interaction of Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases occurring within the same rat liver multienzyme complex are investigated by examining the enzymes catalytic activities and inactivation kinetics. The Michaelis constants for amino acids, ATP and tRNAs of the dissociated aminoacyl-tRNA synthetases are not significantly different from those of the high-Mr multienzyme complex, except in a few cases where the Km values of the dissociated enzymes are higher than those of the high-Mr form. The maximal aminoacylation velocities of the individual aminoacyl-tRNA synthetases are not affected by the presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. Site-specific oxidative modification by ascorbate and nonspecific thermal inactivation of synthetases in the purified rat liver 18 S synthetase complex are examined. Lys- and Arg-tRNA synthetases show remarkably parallel time-courses in both inactivation processes. Leu- and Met-tRNA synthetases also show parallel kinetics in thermal inactivation and possibly oxidative inactivation. Ile-tRNA synthetase shows little inactivation in either process. The oxidative inactivation of Lys- and Arg-tRNA synthetases can be reversed by addition of dithiothreitol. These results suggest that synthetases within the same high-Mr complex catalyze aminoacylation reactions independently; however, the stabilities of some of the synthetases in the multienzyme complex are coupled. In particular, the stability of Arg-tRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase.