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[The tRNA anticodon is recognized by aminoacyl-tRNA-synthetase].

Data concerning the regions of tRNA molecules recognized by cognate aminoacyl-tRNA synthetases and obtained recently by the new methods (the synthesis of mutant tRNA genes and their transcription in vitro, chimeric suppressor tRNA, RNA engineering) are briefly discussed. The results of several laboratories are in full agreement with the hypothesis proposed earlier on the role of a tRNA anticodon as a specific region by which the enzyme identifies various tRNAs. More than half of tRNAs belong to the group recognized by the aminoacyl-tRNA synthetases by this mechanism. The behavior of natural and artificially constructed tRNAs which contain modified anticodons, but conserve amino acid specificity in the reaction of aminoacylation, indicates that in some tRNAs the role of the anticodon in the tRNA identity is minor if any. This group of tRNA is recognized by the aminoacyl-tRNA synthetases predominantly via the double helical region of the molecule. The authors discuss the significance of the absence of topographic conservatism in the mechanism by which tRNAs are recognized by the aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1994

Mechanisms of the transfer of aminoacyl-tRNA from aminoacyl-tRNA synthetase to the elongation factor 1 alpha.

Aspartylation of mammalian tRNAAsp by bacteria-expressed human aspartyl-tRNA synthetase (hDRS) was examined. The kinetics of the aspartylation of tRNA was consistent with the following reaction pathway, [formula: see text] where E, represents aspartyl-tRNA synthetase. A set of rate constants was obtained which fit single turnover time courses at varying concentrations of the enzyme, tRNA, and AMP using the SAAM program. The dissociation of Asp-tRNA (k3) was found to be rate limiting. The elongation factor 1 alpha (EF1 alpha) and GTP stimulated the hDRS aspartylation. The stimulation depended on the presence of both EF1 alpha and GTP. Kinetic analysis indicated that EF1 alpha formed a complex with the hDRS-Asp-tRNA complex and stimulated the dissociation of Asp-tRNA. In the presence of 0.5 M NH4Cl, which enhances the binding of Asp-tRNA by EF1 alpha, hDRS-bound Asp-tRNA can be transferred directly to EF1 alpha. The implications of these results on the function of the multi-tRNA synthetase complex will be discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1995

Molecular recognition of tRNA(Pro) by Escherichia coli proline-tRNA synthetase.

We have investigated the molecular recognition of tRNA(Pro) by Escherichia coli proline tRNA synthetase (ProRS) in vitro using semi-synthetic tRNAs and site-directed mutagenesis of full-length tRNA transcripts. These studies have led to an improved understanding of how this class II synthetase interacts with its tRNA substrate. The ability to efficiently aminoacylate a tRNA(Pro) molecule assembled by annealing together a shorter, chemically synthesized oligonucleotide and a 3/4 tRNA prepared enzymatically, has facilitated the identification of RNA structural features that are critical for aminoacylation by ProRS. This approach has been successful using either a 3'-3/4 tRNA annealed to a 5'-oligonucleotide or a 5'-3/4 tRNA annealed to a 3'-oligonucleotide. These studies show that ProRS appears to be particularly sensitive to mutations that result in structural changes in the core region of tRNA(Pro). Moreover, the so-called "variable pocket" nucleotides appear to be dispensable for aminoacylation. We have also identified a specific 2'-hydroxyl-base interaction between the ribose of U8 and the 2-amino group of G46 that makes a thermodynamically significant contribution to tRNA(Pro) aminoacylation by E. coli ProRS.

Amino Acyl-tRNA Synthetases↗
PubMed1980

Valyl-tRNA and leucyl-tRNA synthetases in wheat germ and seedlings.

Valyl- and leucyl-tRNA synthetases (Val-RS and Leu-RS) isolated from wheat germ and seedlings were separated by chromatography on hydroxylapatite into organellar (Val-RS I and Leu-RS I) and cytoplasmic (Val-RS II and Leu-RS II) enzymes; the enzyme extracted from isolated chloroplasts and mitochondria corresponded to the RS I fractions. It was proved by RPC-5 chromatography of tRNA Val that Val-RS I and Val-RS II recognized all five isoacceptor tRNA Val species both from wheat germ and leaves, as well as tRNA Val from E. coli. However, out of the six isoacceptor tRNA Leu species, Leu-RS II aminoacylated two cytoplasmic species only, while Leu-RS I, the remaining four organellar tRNA Leu fractions. Both leucyl-tRNA synthetases charged E. coli tRNA, Leu-RS I more effectively than Leu-RS II. The absence of fraction RS I in cytosol seems to indicate that valyl- and leucyl-tRNA synthetases (coded for by the nuclear genome) were modified to the organellar forms after (or during) passage into organelles.

Amino Acyl-tRNA Synthetases↗
PubMed1994

tRNA structure and ribosomal function. I. tRNA nucleotide 27-43 mutations enhance first position wobble.

Transfer RNA su7 G36 is a derivative of tRNA(Trp) with a 3'GUC anticodon complementary to the glutamine codon CAG. This tRNA requires a normally forbidden G-U wobble at the first codon position to suppress a UAG (amber) termination codon. Measurement of amber suppression by mutated su7 G36 tRNAs and correction for tRNA levels and aminoacylation allowed calculation of KUAG, a linearized index of in vivo ribosomal function. Following saturating mutagenesis of the anticodon arm of su7 G36, screening for UAG suppression using a lacZ reporter yielded tRNAs with up to 40-fold increased first position G-U wobble, judged from KUAG. The parental anticodon helix has minimized this type of miscoding, and virtually all changes in the top base-pair of the anticodon helix, nucleotides (nt) 27-43, increased the error. Thus, misincorporation of amino acids due to aberrant first position wobble is apparently prevented by normal tRNA structure, which is specifically altered by substitution at nt 27-43, the top base-pair of the anticodon helix. All 16 permutations of nt 27-43, the hotspot for increased wobble, were subsequently constructed and compared. Comparison of values for tRNA coding function, tRNA level, and aminoacylation for the 16 suggest that a tRNA conformational change, specifically involving both nt 27-43, differentially affects all these tRNA functions. This conformational alteration, which presumably occurs normally on the ribosome, appears more complex than simple breakage of the normal 27-43 base-pair. We suggest that the change is in the angle and/or flexibility of the tRNA L-shape. Among these 16 tRNAs, efficient wobble is strongly and inversely correlated with good aminoacylation and high tRNA levels; this quality may have been selected. Constraints on the sequences of natural tRNAs suggest that nt 27-43 have effects on function in many tRNAs.

Anticodon↗
PubMed1997

Selenocysteine tRNA[Ser]Sec levels and selenium-dependent glutathione peroxidase activity in mouse embryonic stem cells heterozygous for a targeted mutation in the tRNA[Ser]Sec gene.

To investigate the effect of a reduced level of selenocysteine (Sec) tRNA[Ser]Sec in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA[Ser]Sec gene. The presence of a single functional gene in ES cells afforded us an opportunity to determine directly in the cell line the effect of reduced gene dosage on (1) the levels of the Sec tRNA[Ser]Sec population, (2) the distributions of the isoacceptors within the Sec tRNA population, and (3) selenoprotein biosynthesis. We therefore determined the amounts and distributions of the two major tRNA[Ser]Sec isoacceptors, designated mcm5U and mcm5Um, within the Sec tRNA population and determined the activity of the anti-oxidant, selenium-containing glutathione peroxidase (GPx) in the heterozygotes and in wild type cells grown in media with and without added selenium. The level of the Sec tRNA[Ser]Sec population in the heterozygotes was approximately 60% of that of wild type cells grown in media under normal conditions, while the ratio of the mcmU isoacceptor in wild type vs mutant cells was approximately 2:1 and of the mcmUm isoacceptor approximately 1:1. In the presence of media supplemented with selenium, the Sec tRNA[Ser]Sec population increased about 20% in wild type cells and virtually not all in heterozygous cells, and the level of the Sec tRNA[Ser]Sec population was, therefore, approximately 50% of that of wild type cells. GPx activity was indistinguishable among these cell lines in either selenium-supplemented or unsupplemented media, indicating that the resultant changes in tRNA[Ser]Sec levels did not have a measurable effect on GPx biosynthesis.

Animals↗
PubMed2004

The nuclear tRNA aminoacylation-dependent pathway may be the principal route used to export tRNA from the nucleus in Saccharomyces cerevisiae.

Nuclear tRNA export in Saccharomyces cerevisiae has been proposed to involve three pathways, designated Los1p-dependent, Los1p-independent nuclear aminoacylation-dependent, and Los1p- and nuclear aminoacylation-independent. Here, a comprehensive biochemical analysis was performed to identify tRNAs exported by the aminoacylation-dependent and -independent pathways of S. cerevisiae. Interestingly, the major tRNA species of at least 19 families were found in the aminoacylated form in the nucleus. tRNAs known to be exported by the export receptor Los1p were also aminoacylated in the nucleus of both wild-type and mutant Los1p strains. FISH (fluorescence in situ hybridization) analyses showed that tRNA(Tyr) co-localizes with the U18 small nucleolar RNA in the nucleolus of a tyrosyl-tRNA synthetase mutant strain defective in nuclear tRNA(Tyr) export because of a block in nuclear tRNA(Tyr) aminoacylation. tRNA(Tyr) was also found in the nucleolus of a utp8 mutant strain defective in nuclear tRNA export but not nuclear tRNA aminoacylation. These results strongly suggest that the nuclear aminoacylation-dependent pathway is principally responsible for tRNA export in S. cerevisiae and that Los1p is an export receptor of this pathway. It is also likely that in mammalian cells tRNAs are mainly exported from the nucleus by the nuclear aminoacylation-dependent pathway. In addition, the data are consistent with the idea that nuclear aminoacylation is used as a quality control mechanism for ensuring nuclear export of only mature and functional tRNAs, and that this quality assurance step occurs in the nucleolus.

Active Transport, Cell Nucleus↗
PubMed2002

Nested evolution of a tRNA(Leu)(UAA) group I intron by both horizontal intron transfer and recombination of the entire tRNA locus.

The origin and evolution of bacterial introns are still controversial issues. Here we present data on the distribution and evolution of a recently discovered divergent tRNA(Leu)(UAA) intron. The intron shows a higher sequence affiliation with introns in tRNA(Ile)(CAU) and tRNA(Arg)(CCU) genes in alpha- and beta-proteobacteria, respectively, than with other cyanobacterial tRNA(Leu)(UAA) group I introns. The divergent tRNA(Leu)(UAA) intron is sporadically distributed both within the Nostoc and the Microcystis radiations. The complete tRNA gene, including flanking regions and intron from Microcystis aeruginosa strain NIVA-CYA 57, was sequenced in order to elucidate the evolutionary pattern of this intron. Phylogenetic reconstruction gave statistical evidence for different phylogenies for the intron and exon sequences, supporting an evolutionary model involving horizontal intron transfer. The distribution of the tRNA gene, its flanking regions, and the introns were addressed by Southern hybridization and PCR amplification. The tRNA gene, including the flanking regions, were absent in the intronless stains but present in the intron-containing strains. This suggests that the sporadic distribution of this intron within the Microcystis genus cannot be attributed to intron mobility but rather to an instability of the entire tRNA(Leu)(UAA) intron-containing genome region. Taken together, the complete data set for the evolution of this intron can best be explained by a model involving a nested evolution of the intron, i.e., wherein the intron has been transferred horizontally (probably through a single or a few events) to a tRNA(Leu)(UAA) gene which is located within a unstable genome region.

Base Sequence↗
PubMed2002

Is tRNA binding or tRNA mimicry mandatory for translation factors?

tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.

Molecular Mimicry↗
PubMed1992

The tRNA species for redundant genetic codons NNU and NNC. A thought on the absence of phenylalanine tRNA with AAA anticodon in Escherichia coli.

The redundant genetic codons NNU and NNC (where N is A, T, G, or C) specify the same amino acid and are decoded by their cognate tRNAs, which contain either a guanosine or a modified base in the wobble position of the anticodons. Since tRNAs with an adenosine in the wobble position of the anticodon, which are complementary to the NNU codons, are not found naturally, we have generated a tRNA(Phe) with AAA anticodon and examined how an adenosine in the wobble position would affect its biological function in Escherichia coli. We found that the tRNA(Phe) with GAA anticodon (wild-type) repressed the expression of the pheA gene via tRNA(Phe)-mediated attenuation of transcription, whereas the tRNA(Phe) with AAA anticodon did not influence the expression of the pheA gene. Furthermore, elevated levels of tRNA(Phe)(AAA) did not support the growth of an E. coli strain carrying a temperature-sensitive mutation in the pheS gene at 42 degrees C. Since the presence of a multicopy plasmid carrying the gene that encodes tRNA(Phe)(GAA), a substrate for phenylalanyl tRNA synthetase, enables the E. coli strain carrying the pheS(Ts) mutation to grow at 42 degrees C, the above observation suggests that unlike tRNA(Phe)(GAA), tRNA(Phe)(AAA) is not a good substrate for phenylalanyl-tRNA synthetase. Therefore, we postulate that the presence of adenosine at the wobble position of anticodons was specifically eliminated and the tRNAs with guanosine or a modified base in the wobble position were selected to decode both NNU and NNC codons in E. coli.

Anticodon↗
PubMed1988

Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons. I. The tRNA gene cluster of rrnB has an internal promoter.

Although the sequence and organization of many Bacillus subtilis tRNA genes are known, primary transcripts from these regions have not been previously analyzed. In this paper, S1 nuclease mapping, S1-type mapping, and Northern analyses were applied to the end of the 23 S rRNA, the 5 S rRNA, and the 21 tRNA genes of B. subtilis operon rrnB. Primary transcripts from the 5 S rRNA and tRNA genes up to approximately 600-800 nucleotides long were observed with S1-type mapping. The presence of discrete bands of processing intermediates indicated preferred processing points within the initial transcript. S1 nuclease mapping delineated a start point for transcription between the second and third tRNA genes. The -10 sequence was within the 37-base pair spacer region between tRNA genes, and the -35 sequence was within the structural gene for the upstream tRNA. Precursors from this region were evident during midexponential growth and two sporulation stages. Thus, in addition to promotion from the rRNA promoters, 19 of the 21 downstream tRNA genes are also under the control of an internal tRNA gene promoter. The accompanying paper (Vold, B. S., Green, C. J., Narasimhan, N., Strem, M., and Hansen, J. N. (1988) J. Biol. Chem. 263, 14485-14490) investigates the minor 5 S rRNA and 16 tRNA genes of another rRNA-tRNA gene set and emphasizes unique promoter elements in that system as well as a potentially unique rRNA processing scheme.

Base Sequence↗
PubMed1981

[tRNA- and aminoacyl-tRNA in the rabbit liver in ontogenesis (author's transl)].

The acceptor capacity of tRNAs and tRNA-synthetases activities of liver extracts was compared for rabbits of different age groups. The level of maximum tRNA aminoacylation in vitro of newborn rabbit (3-5 days) liver for glycine, glutamic acid, leucine, tryptophan, and phenylalanine and tRNA of old rabbits (5 years) liver for glutamic acid, leucine, phenylalanine, and glycine was 1,5-3 times lesser in comparison to that of 1 and 2 years rabbits. The pool of endogenous aminoacyl-tRNAs in 3-5 days rabbits liver was also considerably lower for above mentioned amino acids. The results of short-term heating of tRNA preparations in the presence of Mg2- given the possibility to assume that the decreases tRNA preparations in the presence of Mg2+ give the possibility to assume that the decreases tRNA acceptor activity of the newborn animals depends upon the presence of tRNA inactive conformers. Another reason is the greater loss of the CCA-end in mentioned tRNA. None of these factors in the reason of low acceptor activity of old rabbit liver tRNA which seems to be caused by more considerable disturbances in he molecular structure. Aminoacyl-tRNA-synthetases activities of liver extracts of the same age groups increases during development of animals and decrease subsequently in the liver of old (5 years) rabbits. However, dynamics and quantitative characteristics of these changes are not similar for enzymes of different amino acid specificities.

Aging↗
PubMed1980

Dissection of the active site of rabbit liver tRNA nucleotidyltransferase. Specificity and properties of the tRNA and acceptor subsites determined with model acceptor substrates.

The specificity of rabbit liver tRNA nucleotidyltransferase with respect to its interaction with acceptor residues at the 3' end of tRNA was analyzed using a model acceptor system consisting of dinucleoside monophosphates or nucleosides. Of all the dinucleoside monophosphates tested, only CpC was an active AMP acceptor, indicating that the specificity of the enzyme conforms exactly to the structure present at the 3' terminus of the natural acceptor, tRNA-C-C. Similarly, CMP incorporation into model acceptors closely paralleled the specificity seen with tRNA-C and tRNA-X. Competition studies between the model acceptors and tRNAs with modified 3' termini suggested that the model compounds bind to the enzyme at the site normally recognizing the 3' terminus of tRNA. Comparison of nucleotide incorporation into tRNAs and into the model acceptors revealed a number of differences which allowed us to separate effects on tRNA structure from direct effects on the reaction. These studies enabled us to distinguish several subsites on the enzyme: an ATP-donor site, two sites specifically recognizing the 2 terminal C residues on tRNA, and a site recognizing the nonreacting part of the tRNA. Thus, these results support several features of the multisite model previously proposed (Deutscher, M. P. (1972) J. Biol. Chem. 247, 459-468) to explain tRNA nucleotidyltransferase action.

Adenosine Monophosphate↗
PubMed1997

Unconventional structure of tRNA(Lys)SUU anticodon explains tRNA's role in bacterial and mammalian ribosomal frameshifting and primer selection by HIV-1.

Transfer RNA(Lys)SUU, with a 5-modified-2-thiouridine at wobble position 34, facilitates -1 frameshifts for correct translation of the Escherichia coli DNA polymerase gamma subunit and retroviral polymerases. Peptidyl-tRNA(Lys)SUU prematurely terminates translation more often than other tRNAs. In order to determine if the anticodon structures of bacterial and mammalian tRNA(Lys)SUU species explain these observations, oligonucleotides corresponding to the anticodon regions of mammalian and E. coli tRNA(Lys)SUU were synthesized and their physicochemical properties compared with that of E. coli tRNA(Glu)SUC. The anticodon region of tRNA(Lys)SUU was stabilized by an unusual interaction between the side chains of the 5-modified-s(2)U34 and N-6-threonylcarbamoyl-adenosine-37 (t(6)A37), a combination of modified nucleosides unique to tRNA(Lys)SUU species. This first observation of modified nucleoside side-chain interactions is analogous to the interactions of amino acid side chains in proteins. The tRNA(Lys)SUU anticodon structure was determined from NMR restraints on model oligonucleotides. With only two of three anticodon bases available for codon pairing, this unconventional anticodon structure is a reasonable explanation for the bacterial and mammalian tRNA(Lys)SUU tendency to frameshift. A two-out-of-three reading of coding triplets also explains the increased rate at which peptidyl-tRNA(Lys)SUU prematurely terminates translation. In addition, modified nucleoside interaction distorts the anticodon loop. The distorted loop is a possible structural determinant for the preferential selection of tRNA(Lys3)SUU as primer of HIV-1 reverse transcriptase in vivo.

Anticodon↗
PubMed1998

tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases.

Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension. In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other. From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding. Phe85 and Gln96 play a critical role in this spatial organization. This scaffold can recognize directly U35 at the center of the anticodon. Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36. The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45). Additional free energy of binding is recovered from the resulting network of cooperative interactions. Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37). In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop. In addition, Glu135 would repulse a cytosine base at position 35. Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases. The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.

Acylation↗
PubMed1985

Association of an aminoacyl-tRNA synthetase complex and of phenylalanyl-tRNA synthetase with the cytoskeletal framework fraction from mammalian cells.

The intracellular distribution of several mammalian aminoacyl-tRNA synthetases was investigated by biochemical and immunocytological approaches. The fraction of amino-acyl-tRNA synthetases bound to the detergent-insoluble cytoskeletal framework obtained after extraction of NRK cells by 0.1% Triton X-100 was estimated, by activity measurements, to about 80% for phenylalanyl-tRNA synthetase and 40% for the high-molecular-weight (HMW) complex containing the seven aminoacyl-tRNA synthetases specific for glutamic acid, isoleucine, leucine, methionine, glutamine, lysine, and arginine. This association was shown to be salt-dependent. The subcellular localization of these enzymes was examined using an immunocytological approach. When cultured cells were fixed with paraformaldehyde and then permeabilized with Triton X-100, a fairly uniform cytoplasmic labelling was observed with antibodies directed to the aminoacyl-tRNA synthetase complex or to phenylalanyl-tRNA synthetase. By contrast, when cells were extracted with 0.1% Triton X-100 prior to fixation with paraformaldehyde, the staining patterns obtained with antibodies to aminoacyl-tRNA synthetases were very similar to that obtained with antibodies to rough endoplasmic reticulum, as assessed by single or double indirect immunofluorescence microscopy. These results suggest that free and bound forms of these aminoacyl-tRNA synthetases may coexist within the cell. In addition to cytoplasmic labelling, antibodies directed to phenylalanyl-tRNA synthetase stained the nucleus of rapidly growing cells. The possible significance of this finding is discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1988

Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNA(Trp) aminoacylation.

Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.47) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) is kt = 33 +/- 3 S-1. When starting the aminoacylation reaction with a mono(tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(tryptophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1990

Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for protein synthesis.

Arginyl-tRNA synthetase (arginine-tRNA ligase, EC 6.1.1.19) is found in extracts of mammalian cells both as a free protein (Mr = 60,000) and as a component (Mr approximately 72,000) of the high molecular weight aminoacyl-tRNA synthetase complex (Mr greater than 10(6). Several pieces of evidence indicate that the low molecular weight free form is not a proteolytic degradation product of the complex-bound enzyme but that it preexists in vivo: (i) the endogenous free form differs in size from the active proteolytic fragment generated in vitro, (ii) conditions expected to increase or decrease the amount of proteolysis do not alter the ratio of the two forms of the enzyme, and (iii) the free form contains an NH2-terminal methionine residue. A model is presented that provides a rationale for the existence of two forms of arginyl-tRNA synthetase in cells. In this model the complexed enzyme supplies arginyl-tRNA for protein synthesis, whereas the free enzyme provides arginyl-tRNA for the NH2-terminal arginine modification of proteins by arginyl-tRNA:protein arginyltransferase. This latter process targets certain proteins for removal by the ubiquitin-dependent protein degradation pathway. The necessity for an additional pool of arginyl-tRNA for the modification reaction leads to the conclusion that the arginyl-tRNA destined for protein synthesis (and/or protein modification) is channeled and unavailable for other processes. Other evidence supporting channeling in protein synthesis is discussed.

Amino Acid Sequence↗