Reversible inactivation of tRNA nucleotidyltransferase from baker's yeast by tRNAPhe containing iodoacetamide-alkylated 2-thiocytidine in normal and additional positions.

2-Thiocytidine 5'-triphosphate, s2CTP, is able to replace CTP as a substrate for tRNA nucleotidyltransferase. s2CMP can be incorporated into both cytidine sites of the C-C-A terminus common to all tRNAs, and in the absence of ATP into at least two additional positions. This was shown by alkylation of the 2-thiocytidine residues with iodo[14C]acetamide, total nucleoside analysis, microgel electrophoresis and analysis of RNase T1 fragments of these tRNAs. The incorporation of the 3'-terminal AMP is not influenced by the additional s2CMP residues at pH 9.0. However, at pH 7.6 the additional s2CMP residues are hydrolysed and AMP can be incorporated into the normal position. Two different tRNAs with terminal 2-thiocytidine alkylated by iodoacetamide inhibit tRNA nucleotidyltransferase. This inhibition is significantly slower if an elongated species is used compared to a tRNA with alkylated 2-thiocytidine in the normal position 75. The addition of 2-mercaptoethanol reactivates the enzyme and leads to a cytidine containing tRNA. This reaction identifies the attacking nucleophile of the enzyme as cysteine residue, which is probably identical to a cysteine residue found in a similar experiment reported previously. The mechanism of the enzymatic and chemical reactions is discussed.     

Purification and characterization of a mutant tRNA nucleotidyltransferase

tRNA nucleotidyltransferase has been extensively purified from a mutant strain of Escherichia coli which displays greatly decreased AMP incorporation, but normal CMP incorporation. The defect in AMP incorporation is retained throughout the purification of the mutant protein. The mutant protein behaves identically to the wild-type protein with regard to elution position on various chromatographic columns, and both have similar molecular weights of about 50000. The defect in the mutant protein is accentuated by the use of yeast tRNA rather than E. coli tRNA-C--C as substrate, by decreased pH, by increased ionic strength and by decreased divalent cation concentration. Substitution of MN2+ for Mg2+ greatly increases the relative activity of the mutant enzyme. In all these cases, CMP incorporation by the mutant enzyme remains the same as the wild-type enzyme. The Km values of the mutant enzyme for its tRNA and triphosphate substrates are unchanged, and the mutant protein is as stable as the wild type with respect to temperature inactivation. These results strongly suggest that the mutation is in the structural gene for tRNA nucleotidyltransferase, and that the mutation probably does not affect the overall structure of the mutant protein, but only a localized region near the AMP-incorporating site.     

Isolation and properties of tRNA nucleotidyltransferase from wheat embryos.

From wheat embryos, tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated. By chromatography on Sepharose 6B, DEAE-cellulose and affinity chromatography on tRNA-hydrazyl-Sepharose 4B, 7000-fold purification of the enzyme was achieved. The enzyme required for its activity Mg2+ or Mn2+ ion. ATP inhibited incorporation of CMP from CTP into lupin tRNA, and CTP acted as a competitive inhibitor of AMP incorporation from ATP. The regulatory role of ATP in incorporation of terminal CMP into tRNA is discussed. The incorporation of terminal CMP into tRNA deprived of terminal CCA or CA, was also studied.     

A novel nucleolytic activity associated with rabbit liver tRNA nucleotidyltransferase

Purified preparations of rabbit liver tRNA nucleotidyltransferase contain a nucleolytic activity which removes terminal CMP residues from tRNA-C-C and tRNA-C-C-C. Other tRNA molecules, such as tRNA-C-C-A, tRNA-C-A, tRNA-C-U and tRNA-C are not substrates for this reaction. The activity exhibits a sharp optimum at about pH 10 and a divalent cation (Mg⁺⁺ or Mn⁺⁺) is required. The reaction is inhibited by ATP, CTP, pyrophosphate and potassium chloride. The relation of this activity to other reactions catalyzed by tRNA nucleotidyltransferase is discussed.     

Study of the Escherichia coli tRNA nucleotidyltransferase. Specificity of the enzyme for nucleoside triphosphates

Besides the main reactions leading to the repair of tRNA molecules deprived of part or all of their 3′ terminal -pCpCpA sequence, purified E. coli tRNA nucleotidyltransferase catalyzes in vitro, under certain conditions the synthesis of sequences not found in natural tRNAs. In the absence of CTP, AMP is incorporated directly into tRNA-pX or tRNA-pXpC leading to tRNA-pXpA or tRNA-pXpCpA respectively. In the absence of ATP one extra CMP is added to tRNA-pXpCpC to form tRNA-pXpCpCpC. UMP can be incorporated instead of CMP and the sequence -pXpU and -pXpCpU formed. The incorporation of UMP cannot be followed by the incorporation of either a second UMP or an AMP. In all cases, the rate of misincorporation is lower than the rate of the synthesis of the normal sequence.The apparent K_M of the enzyme for UTP is 3.0 10⁻⁴ M. CTP inhibits competitively the incorporation of UMP into tRNA-pX with a K_i value (1.6 10⁻⁵ M) close to its apparent K_M.     

Kinetic mechanism of tRNA nucleotidyltransferase from Escherichia coli.

A kinetic analysis of the incorporation of AMP into tRNA lacking the 3'-terminal residue by tRNA nucleotidyltransferase (EC 2.2.7.25) from Escherichia coli is presented. Initial velocity studies demonstrate that the mechanism is sequential and that high concentrations of tRNA give rise to substrate inhibition which is noncompetitive with respect to ATP. In addition, the substrate inhibition is more pronounced in the presence of pyrophosphate, which suggests the formation of an inhibitory enzyme-pyrophosphate-tRNA complex. Noncompetitive product inhibition is observed between all possible pairs of substrates and products. ADP and alpha,beta-methylene adenosine triphosphate are competitive dead end inhibitors of ATP, while the latter is a noncompetitive dead end inhibitor of the tRNA substrate. A nonrapid equilibrium random mechanism is proposed which is consistent with these data and offers an explanation for the noncompetitive substrate inhibition by tRNA.     

Limited hydrolysis of tRNA by phosphodiesterase.

Digestion of tRNA by electrophoretically pure phosphodiesterase is limited to a short sequence of nucleotides at the 3'-terminus. On the average, four percent of all nucleotides can be released from tRNA. The optimum Mg2 concentration is 10mM and the optimum pH 9.2. The mode of action is a random attack by the enzyme on the substrate. The terminal AMP is completely removed at 15 degrees C after short incubation; about 400 mol of AMP were removed per min by 1 mol of enzyme. The following CMP residues are released much more slowly; at 15 degrees C incompletely, and at 37 degrees C more or less completely in 1 h. In about 50% of the tRNA molecules, the fourth nucleotide could be removed in very long incubations or with very high enzyme concentrations.     

Purification of a cyclic nucleotide phosphodiesterase from bovine brain using blue dextran-Sepharose chromatography.

The soluble high Km form of cyclic nucleotide phosphodiesterase (EC 3.4.1.17) was purified over 2000-fold from bovine brain homogenates principally using blue dextran-Sepharose chromatography. The purified protein has a specific enzymic activity of 167 units/mg and appears homogeneous when examined by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 1.26 +/- 0.05 x 10(5) consisting of two apparently identical polypeptide chains. Kinetic measurements indicate that the substrates cyclic GMP and cyclic AMP each have a single Km value, 9 +/- 1 micron and 150 +/- 50 micron, respectively, that the two cyclic nucleotides compete for the same catalytic site, that the blue dye of blue dextran-Sepharose is a competitive inhibitor for the cyclic nucleotides, and that the Vmax with cyclic AMP as substrate is about an order of magnitude larger than that for cyclic GMP. Bovine brain calmodulin stimulates the catalytic rate of the purified enzyme in the presence of Ca2+ by increasing the Vmax associated with each cyclic nucleotide substrate.     

CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?

The CCA-adding enzyme repairs the 3''-terminal CCA sequence of all tRNAs. To determine how the enzyme recognizes tRNA, we probed critical contacts between tRNA substrates and the archaeal Sulfolobus shibatae class I and the eubacterial Escherichia coli class II CCA-adding enzymes. Both CTP addition to tRNA-C and ATP addition to tRNA-CC were dramatically inhibited by alkylation of the same tRNA phosphates in the acceptor stem and TPsiC stem-loop. Both enzymes also protected the same tRNA phosphates in tRNA-C and tRNA-CC. Thus the tRNA substrate must remain fixed on the enzyme surface during CA addition. Indeed, tRNA-C cross-linked to the S. shibatae enzyme remains fully active for addition of CTP and ATP. We propose that the growing 3''-terminus of the tRNA progressively refolds to allow the solitary active site to reuse a single CTP binding site. The ATP binding site would then be created collaboratively by the refolded CC terminus and the enzyme, and nucleotide addition would cease when the nucleotide binding pocket is full. The template for CCA addition would be a dynamic ribonucleoprotein structure.     

Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition

The kinetic properties of highly purified human placental cytoplasmic 5'-nucleotidase were investigated. Initial velocity studies gave Michaelis constants for AMP, IMP, and CMP of 18, 30, and 2.2 microM, respectively. The enzyme shows the following relative Vmax values: CMP greater than UMP greater than dUMP greater than GMP greater than AMP greater than dCMP greater than IMP. The activity was magnesium-dependent, and this cation binds sequentially with a Km of 14 microM for AMP and an apparent Km of 6 mM for magnesium. A large variety of purine, pyrimidine, and pyridine compounds exert an inhibitory effect on enzyme activity. IMP, GMP, and NADH produce almost 100% inhibition at 1.0 mM. Nucleoside di- and triphosphates are potent inhibitors. ATP and ADP are competitive inhibitors with respect to AMP and IMP as substrates with Ki values of 100 and 15 microM, respectively. Inorganic phosphate is a noncompetitive inhibitor with Ki values of 19 and 43 mM. Nucleosides and other compounds studied produce only a modest decrease of enzyme activity at 1 mM. Our findings suggest that the enzyme is regulated under physiological conditions by the concentrations of magnesium, nucleoside 5'-monophosphates, and nucleoside di- and triphosphates. The nucleotide pool concentration regulates the enzyme possibly by a mechanism of heterogeneous metabolic pool inhibition. These properties of human placental cytoplasmic 5'-nucleotidase may be related to the control of nucleotide degradation in vivo.     

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.     

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.     

Effects of spermine and magnesium ions on the aminoacylation of yeast tRNA(Tyr)

Stimulatory effects of Mg2+ and spermine on the kinetics of the aminoacylation of tRNA(Tyr) were examined using purified yeast tRNA(Tyr) and tyrosyl-tRNA synthetase. The apparent Km for tRNA(Tyr) was the lowest at Mg2+ concentrations between 2 and 5 mM and was not influenced by spermine. In the absence of spermine, the apparent Vmax was the highest at Mg2+ concentrations of 5 mM or higher, whereas the presence of spermine strongly stimulated the reaction at lower Mg2+ concentrations. Spermine alone could not substitute for Mg2+, nor was it able, at any Mg2+ concentration, to increase the reaction rate above the level reached at high concentrations of Mg2+ alone. Calculations of the concentration of Mg3.tRNA(Tyr) complex as a function of initial Mg2+ concentration, using the binding constants derived from physical measurements, allow the conclusion that spermine exerts its stimulatory activity by creating strong binding sites for Mg2+; this would enable the tRNA to assume the conformation required for optimal aminoacylation. The conformational requirement for the first tRNA: synthetase encounter is obviously less stringent, since the apparent Km for tRNA(Tyr) is not influenced by spermine.     

Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae. II. Catalytic mechanism.

Yeast histidine tRNA guanylyltransferase (TGT) catalyzes in the presence of ATP the addition of GTP to the 5' end of eukaryotic cytoplasmic tRNAHis species. A study of the enzyme mechanism with purified protein showed that during the first step ATP is cleaved to AMP and PPi creating adenylylated TGT. In a second step the activated enzyme forms a stable complex with its cognate tRNA substrate. The 5'-phosphate of the tRNA is adenylylated by nucleotide transfer from the adenylylated guanylyltransferase to form A(5')pp(5')N at the 5'-end of the tRNA. Finally, the 3'-hydroxyl of GTP adds to the activated 5' terminus of the tRNA with the release of AMP. This mechanism of tRNAHis guanylyltransferase is very similar to that of RNA ligases. dATP can substitute for ATP in this reaction. Since among several guanosine compounds active in this reaction GTP is most efficiently added we believe that it is the natural substrate of TGT.     

Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.

Several enzymes of the glycolytic pathway are phosphorylated in vitro and in vivo by retroviral transforming protein kinases. These substrates include the enzymes phosphoglycerate mutase (PGM), enolase and lactate dehydrogenase (LDH). Here we show that purified EGF (epidermal growth factor)-receptor kinase phosphorylates the enzymes PGM and enolase and also the key regulatory enzymes of the glycolytic pathway, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in an EGF-dependent manner. Stoichiometry of phosphate incorporation into GAPDH (calculated from native Mr) is the highest, reaching approximately 1. LDH and other enzymes of the glycolytic pathway are not phosphorylated by the purified EGF-receptor kinase. These enzymes are phosphorylated under native conditions, and the Km values of EGF-receptor kinase for their phosphorylation are close to the physiological concentrations of these enzymes in the cell. EGF stimulates the reaction by 2-5-fold by increasing the Vmax. without affecting the Km of this process. Phosphorylation is rapid at 22 degrees C and at higher temperatures. However, unlike the self-phosphorylation of EGF-receptor, which occurs at 4 degrees C, the glycolytic enzymes are poorly phosphorylated at this temperature. Some enzymes, in particular enolase, increase the receptor Km for ATP in the autophosphorylation process and thus may act as competitive inhibitors of EGF-receptor self-phosphorylation. On the basis of the Km values of EGF receptor for the substrate enzymes and for ATP in the phosphorylation reaction, these enzymes may also be substrates in vivo for the EGF-receptor kinase.     

Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: identification of amino acid residues labeled by periodate-oxidized tRNA(fMet) molecules having modified lengths at the 3'-acceptor end.

Initiator tRNA molecules modified at the 3'-end and lacking either the A76 (tRNA-C75), the C75-A76 (tRNA-C74), the C74-C75-A76 (tRNA-A73), or the A73-C74-C75-A76 (tRNA-A72) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTST), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C75ox, tRNA-C74ox, tRNA-A73ox, and tRNA-A72ox) abolished both the isotopic [32P]PPi-ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C75ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C75. The peptides of MTST labeled with either tRNA-C75ox or tRNA-C74ox were identified. The chymotryptic digestion of the covalent MTST.[14C]tRNA-C75ox complex yielded four peptides (A-D). In the case of tRNA-C74ox, only two of the above peptides (C and D) were identified. Peptides A, B, C, and D corresponded to fragments Ser334-Phe340, Lys61-Leu65, Val141-Tyr165, and Glu433-Phe437, respectively, in the MTST primary structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutation in Escherichia coli tRNA nucleotidyltransferase that affects only AMP incorporation is in a sequence often associated with nucleotide-binding proteins

Escherichia coli strain 5C15 contains a mutation in the cca gene that decreases AMP incorporation by tRNA nucleotidyltransferase while leaving CMP incorporation unaffected. Earlier studies of the purified mutant enzyme suggested that the mutation was localized to the AMP-incorporating site. In order to analyze this mutation in more detail, the cca gene from strain 5C15 was cloned into plasmid pUC8. Analysis of tRNA nucleotidyltransferase activity in extracts of a strain transformed with this plasmid demonstrated an elevated level of CMP incorporation, but low AMP incorporation, as expected from the properties of the original mutant. Sequence analysis of the mutant cca gene revealed only a single G to A point mutation leading to a glycine to aspartic acid substitution at position 70 of the peptide chain. The amino acid change was localized to one of two Gly-X-Gly-X-X-Gly sequences present in the protein. This sequence has been identified previously near the nucleotide-binding domain of various proteins, but it has not been noted in enzymes that incorporate nucleotide residues. However, other sequences often associated with ATP-binding domains are not found in tRNA nucleotidyltransferase. The implications of these findings for our understanding of nucleotide-binding domains are discussed.     

Isoleucyl-tRNA synthetase from baker's yeast. Influence of substrate concentrations on aminoacylation pathways, discrimination between tRNAIle and tRNAVal, and between isoleucine and valine

The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively. At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNAVal such differences could not be detected. The substrate ATP did not show any change of its Km value as far as the reaction was measurable. Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant. The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated.     

Identification of multiple RNases in Xenopus laevis oocytes and their possible role in tRNA processing.

A survey of RNases in Xenopus laevis oocytes has been carried out to identify potential tRNA-processing enzymes in this system. Using a variety of specific and nonspecific substrates, we have shown that oocytes contain multiple RNases with various specificities. Three activities that could cleave the extraneous residues from the artificial tRNA precursor, tRNA-C-[14C]U-C, to generate a substrate for -C-C-A addition by tRNA nucleotidyltransferase were identified. One of these was a cytoplasmic exonuclease which generated predominantly tRNA-C, whereas the other two were nuclear endonucleases which cleaved the precursor to generate tRNA-N. The possible involvement of these activities in 3' tRNA processing in oocytes is discussed.