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12 recordsLinked to original sources
PubMed1976

Distinct steps in the specific binding of tRNA to aminoacyl-tRNA synthetase. Temperature-jump studies on the serine-specific system from yeast and the tyrosine-specific system from Escherichia coli.

The kinetics of the interaction of tRNASer and seryl-tRNA synthetase from yeast as well as of tRNATyr and tyrosyl-tRNA synthetase from Escherichia coli have been investigated by temperature-jump experiments. It could be shown that complex formation proceeds in two distinct steps. This was demonstrated for both the first and the second binding site. The two-step mechanism was deduced from the characteristic concentration dependence of the relaxation times. Seryl-tRNA synthetase recombines with the first tRNA to form an intermediate complex (kI12, kI21), which is transformed in a fast reaction to the final 1:1 complex (kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 X 10(8) M-1 S-1; kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 x 10(8) M-1 S-1; kI21 = 220 S-1; kI23 = 760 S-1; kI32 = 330 S-1. The 1:1 complex can bind a second tRNA. At pH 7.2 without added salt the rate constants are: KII2 = 0.9 X 10(8) M-1 S-1; kII21 = 270 S-1; kII23 = 120 S-1; kII32 = 1250 S-1. The tyrosine-specific system behaves very similarly to the serine-specific system. Data are given for pH 7.2 (pH 6.0) for the binding of the second tRNA: kII12 = 1 X 10(8) (2.5 X 10(8)) M-1 S-1; kII21 = 470 (170) S-1; kII23 = 150 (530) S-1; kII32 = 1540 (720) S-1. The kinetic results are discussed in terms of their relevance to the recognition process and their relation to the anticooperative binding behaviour of tRNA to synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1979

The stereochemical course of amino acid activation by methionyl- and tyrosyl-tRNA synthetases.

Stereochemical analysis has long been recognised as a powerful tool for elucidating the mechanisms of chemical and enzyme-catalysed reactions. Although much is known about the stereochemical course of reactions at saturated carbon, phosphate and thiophosphate esters whose ligands to phosphorus are also tetrahedrally disposed, are capable in principle of revealing sterochemical information about events at the active site of enzymes that transform such substrates. Nucleotidyl transferases are a group of enzymes which in general selectively use one of the diastereoisomers of a nucleoside 5'(1-thiotriphosphate), such as isomers A and B of adenosine 5'(1-thiotriphosphate), designated ATP alpha S-A and ATP alpha S-B, and allow investigation of the stereochemical course of nucleotidyl transfer. We have developed a simple method based on 31P nuclear magnetic resonance spectroscopy for determining the stereochemical course of these reactions, and using this method show here that the nucleotidyl transfer step in two aminoacyl-tRNA synthetases from Escherichia coli occurs with inversion of configuration at phosphorus. These observations greatly constrain the mechanistic possibilities for these enzymes, and are interpreted most simply as a direct 'in line' transfer from ATP to the amino acid.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Experimental evidence for kinetic proofreading in the aminoacylation of tRNA by synthetase.

The enzymatic aminoacylation of tRNA can be viewed as a means of proofreading either the amino acid or the tRNA or both. We have conducted further experimental tests of kinetic proofreading in discriminating between cognate and noncognate amino acids and tRNAs as follows: (formula: see text). In cases (i) and (ii) the amino acids are proofread, in cases (iii) and (iv) the tRNA is proofread, and in case (v), both the amino acid and the tRNA are proofread. ATP consumed per acylation was 400, 1.5, 40, 25, and 1000, respectively. High ATP/aminoacylation ratios are diagnostic for kinetic proofreading.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Flexibility and rigidity in protein crystals.

There is an increasing body of crystallographic evidence for disorder in parts of the main polypeptide chain of certain proteins. The zymogens of pancreatic serine proteinases and tyrosyl-tRNA synthetase are used as examples. It is suggested that reactivity and specificity are favoured by an appropriate rigid conformation, and that disorder may often be involved in some kind of control function.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Purification and some properties of rat liver tyrosyl-tRNA synthetase.

Rat liver cytoplasmic tyrosine:tRNA ligase (tyrosine:tRNA ligase, EC 6.1.1.1) was purified by ultracentrifugation, DEAE-cellulose chromatography and repeated phosphocellulose chromatography by more than 1500-fold. The molecular weight of the enzyme was approx. 150 000 as determined by Sephadex G-200 gel filtration. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme consisted of two subunits, each of 68 000 daltons. We found the following Km values for the enzyme: 13 micrometer for tyrosine and 1.7 mM for ATP in the ATP:PPi exchange reaction and 13 micrometer for tyrosine, 210 micrometer for ATP and 0.14 micrometer for tRNATyr in the aminoacylation reaction. The rate of tyrosyl-tRNA synthesis was 50-fold lower than that of ATP:PPi exchange. Addition of a saturating amount of tRNA did not affect the rate of ATP:PPi exchange.

Adenosine Triphosphate↗
PubMed1979

Numerical analysis of binding studies: a direct procedure avoiding the pitfalls of a Scatchard analysis of equilibrium data for unknown binding models.

It is shown on theoretical grounds that the straightforward analysis of binding data according to Scatchard may lead to erroneous results, especially when more complicated binding schemes are involved. We have demonstrated this point by presenting Scatchard plots with slight variation of experimental parameters. These inherent difficulties of Scatchard analyses can be avoided by applying a direct procedure. We have developed a program, which compares the measured quantity and the theoretical value directly and which considers the following binding models: (i) independent equivalent binding of n ligands; (ii) independent unequivalent binding of 2 ligands; (iii) positive or negative cooperative binding of 2 ligands. Other binding schemes can easily be implemented. We have used this procedure for the evaluation of equilibrium data on the complex formation of tRNA-Tyr and tyrolyl tRNA synthetase from E. coli in terms of different binding models.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Structure and processing of yeast precursor tRNAs containing intervening sequences.

We have isolated a precursor of yeast tRNATyr and shown that it contains an intervening sequence identical to that found in the gene for tRNATyr. The conformation of pre-tRNATyr is similar to that of mature tRNATyr except for the anticodon loop. The loop is sensitive to endonucleolytic cleavage by S1 nuclease near to the ends of the intervening sequence. This pre-tRNA is functionally inactive as it cannot be aminoacylated and the anticodon is not accessible for hydrogen bonding. A crude nuclear extract from yeast contains an excision-ligase activity which will process pre-tRNATyr into mature tRNATyr.

Anticodon↗
PubMed1978

The nucleoside sequence of tyrosine tRNA from Bacillus stearothermophilus.

The nucleotide sequence of tRNATyr from B. stearothermophilus has been determined: pG-G-A-G-G-G-G-s4U-A-G-C-G-A-A-G-U-Gm-G-C-U-A-A-m1A-C-G-C-G-G-C-G-G-A-C-U-Q-U-A-ms2i6A-A-psi-C-C-G-C-U-C-C-C-U-U-U-G-G-G-U-U-C-G-G-C-G-G-T-psi-C-G-A-A-U-C-C-G-U-C-C-C-C-C-U-C-C-A-C-C-AOH. A combination of classical fingerprinting methods, partial nuclease P1 digestion and two-dimensional homochromatography and a rapid "read off" sequencing gel technique were used to establish the complete nucleotide sequence.

Base Sequence↗
PubMed1979

The binding of tyrosinyl-5'-AMP to tyrosyl-tRNA synthetase (E.coli).

The binding between tyrosyl-tRNA synthetase (E.coli) and the alkylanalogue of the aminoacyladenylate, tyrosinyl-5'-AMP, has been investigated by fluorescence titrations and rapid mixing experiments. Tyrosyl-tRNA synthetase has two equivalent and independent binding sites for tyrosinyl-5'-AMP. The intrinsic binding constant is 4 x 10(7)M-1. The binding sites for tRNATyr and tyrosinyl-5'-AMP are independent of each other, the anticooperative mode of tRNA binding being preserved in the presence of tyrosinyl-5-AMP.

Adenosine Monophosphate↗
PubMed1977

Tyroslyl-tRNA synthetase from baker's yeast. Rapid isolation by affinity elution, molecular weight of the enzyme, and determination of essential sulfhydryl groups.

Tyrosyl-tRNA synthetase (EC 6.1.1.1) has been isolated from baker's yeast with an overall purification factor of more than 5000. After opening the cells, pH 4.8 precipitation, ammonium sulfate fractionation, removal of the nucleic acids with DEAE-cellulose and chromatography on CM-Sephadex, the critical purification step is the elution of the cation-exchanger-bound tyrosyl-tRNA synthetase with tRNATyr. The homogeneous enzyme exhibits a molecular weight of 40 000 as estimated by sedimentation equilibrium centrifugation and dodecylsulfate-gel electrophoresis under reducing and non-reducing conditions. Gel filtration experiments show a molecular weight of about 100 000 indicating the existence of an active dimeric form. The possibility of proteolytic cleavage of the enzyme is excluded. The reaction of tyrosyl-tRNA synthetase with p-chloromercuribenzoate and N-ethylmaleimide reveals two repidly reacting sulfhydryl groups per subunit of molecular weight 40 000, as demonstrated by the inhibition of aminoacylation and the isolation of enzyme-inhibitor complexes. In addition an efficient purification method is described for isolating tRNATyr from soluble ribonucleic acid from baker's yeast in three chromatographic steps in a yield of 28%.

Amino Acyl-tRNA Synthetases↗