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R Giegé

Publications and source records attributed to R Giegé.

158 records · Page 9Linked to original sources
PubMed1977

The yeast aminoacyl-tRNA synthetases. Methodology for their complete or partial purification and comparison of their relative activities under various extraction conditions.

Several fractionation steps are described which can be applied to the partial purification of the 20 aminoacyl-tRNA synthetases from commercial baker's yeast. Comparative experiments performed in the presence or absence of protease inhibitors revealed that some enzymes prepared in the presence of the inhibitor exhibit much higher specific activities than the proteins extracted in the absence of the inhibitor. The methodology reported can be used for the simultaneous preparation of several pure aminoacyl-tRNA synthetases. As examples, the large scale purification of phenylalanyl-and valyl-tRNA synthetases are described.

Amino Acyl-tRNA Synthetases↗
PubMed1977

A neutron investigation of yeast valyl-tRNA synthetase interaction with tRNAs.

A new way of studying RNA-protein complexes, using neutron small angle scattering in solution, is described and was applied in the case of the system, yeast valyl-tRNA synthetase, interacting with its cognate and non cognate yeast tRNAs. It was shown that, when limited amounts of tRNA (either cognate or non cognate) are added to valyl-tRNA synthetase, a complex consisting of two enzyme molecules and one tRNA molecule is first formed. It is subsequently dissociated to a one to one complex when more tRNA is present in the solution. The association curve shows a maximum for a molecular ratio, enzyme over tRNA, equal to 2.

Amino Acyl-tRNA Synthetases↗
PubMed1976

Interpretation of tRNA-mischarging kinetics.

Incorrect tRNA aminoacylation reactions are characterized by very slow reaction rates and by the fact that in most cases they are incomplete. In a previous study some of us explained the incompleteness of the correct aminoacylation reactions of tRNA, which can be encountered under certain experimental conditions (for instance low enzyme concentration or high ionic strength) by an equilibrium between the aminoacylation and the deacylation reactions [J. Bonnet and J.P. Ebel (1972) Eur. J.Biochem. 31, 335-344]. In the present report we bring evidence that the incorrect valylation of yeast tRNAfMet by yeast valyl-tRNA synthetase studied under standard experimental conditions, can also be described by a kinetic rate law including the rate equations of the aminoacylation and of the various deacylation reactions. In particular we show that the incomplete mischarging plateaus reflect the existence of an equilibrium between the valylation reaction on the one hand and the spontaneous and enzymic deacylation of valyl-tRNAfMet and the reverse of the valylation reaction on the other hand. However, when the valyl-tRNA synthetase concentration is not very high the reverse reaction of the amino-acylation does not play a predominant part in the establishment of the plateau. These interpretations have been extended to other mischarging systems: valylation of yeast tRNAPhE by yeast valyl-tRNA synthetase and mischarging of tRNAfMet and tRNA2Val from yeast by yeast phenylalanyl-tRNA synthetase. Unusual mischarging kinetics have been discussed. Furthermore, and as in correct systems, we found that during the mischarging of tRNAfMet one ATP is hydrolyzed per tRNA charged with valine. We conclude that the correct and the incorrect amino-acylation of tRNA behave kinetically in a similar way.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Raman spectra and structure of yeast phenylalanine transfer RNA in the crystalline state and in solution.

Laser Raman spectra of yeast phenylalanine transfer RNA have been obtained in solution and in orthorhombic and hexagonal crystals. So far as one can tell from the spectra, which are identical in the two crystal forms, the molecular structure of the tRNA is not altered by differences in molecular packing in these two unit cells. In addition, the spectra of the two crystal forms show the same characteristic Raman frequencies and intensities as those of the tRNA in aqueous solution. Thus the structure of the tRNA molecule appears to be the same in the crystals and in aqueous solution. From the spectroscopic changes that result when Mg2+ ions are removed from the native tRNA, it is concluded that the removal of Mg2+ produces a partial disordering of the ribophosphate backbone of the molecule and a lowering of its melting temperature. The melting is shown to be a complex process in that the vibrations specific for adenine indicate a slightly lower melting temperature and those specific for guanine a slightly higher melting temperature than that of the ribophosphate backbone.

Binding Sites↗
PubMed1975

Three photo-cross-linked complexes of yeast phenylalanine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases.

Yeast tRNA-Phe has been cross-linked photochemically to three aminoacyl-tRNA synthetases, yeast phenylalanyl-tRNA synthetase, Escherichia coli isoleucyl-tRNA synthetase, and E. coli valyl-tRNA synthetase. The two non-cognate enzymes are known to interact with tRNA-Phe. In each complex, three regions on the tRNA are found to cross-link. Two of these are common to all of the complexes, while the third is unique to each. Thus, the cognate and non-cognate complexes bear considerable similarity to each other in the way in which the respective enzyme orients on tRNA-Phe, a result which was also established for the complexes of E. coli tRNA-Ile (BUDZIK, G.P., LAM, S.M., SCHOEMAKER, H.J.P., and SCHIMMEL, P.R. (1975) J. Biol. Chem. 250, 4433-4439). The common regions include a piece extending from the 5'-side of the acceptor stem to the beginning of the dihydrouridine helix, and a segment running from the 3' side of the extra loop into the TpsiC helix. These two regions overlap with and include some of the homologous bases found in eight tRNAs aminoacylated by yeast phenylalanyl-tRNA synthetase (ROE, B., SIROVER, M., and DUDOCK, B. (1973) Biochemistry 12, 4146-4153). Although well separated in the primary and secondary structure, these two segments are in close proximity in the crystallographic tertiary structure. In two of the complexes, the third cross-linked fragment is near to the two common ones. The picture which emerges is that the enzymes all interact with the general area in which the two helical branches of the L-shaped tertiary structure fuse together, with additional interactions on other parts of the tRNAas well.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Complete purification and studies on the structural and kinetic properties of two forms of yeast valyl-tRNA synthetase.

Two forms of baker's yease valyl-tRNA synthetase have been purified to apparent homogeneity by classical methods. It was demonstrated that one of the two forms of the enzyme originates from the other by proteolysis, the respective amounts of each form depending on the physiological state of the yeast. The species mainly isolated from exponential growing yeast cells is a monomer of 130,000 daltons molecular weight. In stationary phase cells or in commercial yeast the major species is a degraded monomer of 120,000 daltons molecular weight ; however when the purification is carried out in the presence of phenylmethyl-sulphonyl fluoride, or diisopropylfluorophosphate large amounts of the not - degreded monomer can be obtained. Of great practical usefulness is the fact that large amounts of the native enzyme can be obtained pure after only two chromatographic steps on DEAE-cellulose and hydroxylapatite. The kinetic constants for valine, ATP and tRNAVal were determined, as well as the optimum aminoacylation conditions. It was found that the specific activity of the nondegraded valyl-tRNA synthetase is higher than that of the proteolysed enzyme for the aminoacylation reaction. On the contrary, both forms have the same ATP-pyroposphate exchange activity. The amino acids composition of the native enzyme was established. The tryptic fingerprints of the two valyl-tRNA synthetases were studied. Essentially similar maps were obtained. The number of the spots in the fingerprints indicates that the enzymes contain a high proportion of repeated sequences.

Adenosine Triphosphate↗
PubMedDate not supplied

Exploring the aminoacylation function of transfer RNA by macromolecular engineering approaches. Involvement of conformational features in the charging process of yeast tRNA(Asp).

This report presents the conceptual and methodological framework that presently underlies the experiments designed to decipher the structural features in tRNA important for its aminoacylation by aminoacyl-tRNA synthetases. It emphasizes the importance of conformational features in tRNA for an optimized aminoacylation. This is illustrated by selected examples on yeast tRNA(Asp). Using the phage T7 transcriptional system, a series of tRNA(Asp) variants were created in which conformational elements were modified. It is shown that aspartyl-tRNA synthetase tolerates conformational variability in tRNA(Asp) at the level of the D-loop and variable region, of the tertiary Levitt base-pair 15-48 which can be inverted and in the T-arm in which residue 49 can be excised. However, changing the anticodon region completely abolishes the aspartylation capacity of the variants. Transplanting the phenylalanine identity elements into a different tRNA(Asp) variant presenting conformational characteristics of tRNA(Phe) converts this molecule into a phenylalanine acceptor but is less efficient than wild-type tRNA(Phe). This engineered tRNA completely loses its aspartylation capacity, showing that some aspartic acid and phenylalanine identity determinants overlap. The fact that chimeric tRNA(Asp) molecules with altered anticodon regions lose their aspartylation capacity demonstrates that this region is part of the aspartic acid identity of tRNA(Asp).

Aspartate-tRNA Ligase↗