Franco Fasiolo

Ribosome assembly in eukaryotes.

Ribosome synthesis is a highly complex and coordinated process that occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells. Based on the protein composition of several ribosomal subunit precursors recently characterized in yeast, a total of more than 170 factors are predicted to participate in ribosome biogenesis and the list is still growing. So far the majority of ribosomal factors have been implicated in RNA maturation (nucleotide modification and processing). Recent advances gave insight into the process of ribosome export and assembly. Proteomic approaches have provided the first indications for a ribosome assembly pathway in eukaryotes and confirmed the dynamic character of the whole process.

Factors determining the specificity of the tRNA aminoacylation reaction: Non-absolute specificity of tRNA-aminoacyl-tRNA synthetase recognition and particular importance of the maximal velocity

Summary It is generally believed that the specificity of tRNA aminoacylation results solely from a specific recognition between the aminoacyl-tRNA synthetase and the cognate tRNA. In fact, this specificity is not absolute: this is supported by the following observations (1) the existence of tRNA mischarging in homologous systems under usual aminoacylation conditions, (2) the existence of inhibitions produced by « non-cognatetRNA species in correct aminoacylation reactions, (3) the lack of specificity of AMP- and PPi- independent aminoacyl-tRNA synthetase catalysed deacylation of aminoacyl-tRNA species, (4) the isolation of complexes between aminoacyl-tRNA synthetases and non-cognate tRNA species. The affinities between aminoacyl-tRNA synthetases and non-cognate tRNA species, estimated by the Km measurements in mischarging reactions, have been found only diminished by 1 or 2 orders of magnitude as compared to the values found in specific systems, whereas the Vmax values for mischarging have been found diminished by 3 or 4 orders of magnitude. This suggests that tRNA aminoacylation depends more upon the maximal velocity of the reaction than upon the recognition between aminoacyl-tRNA synthetase and tRNA. Furthermore, we found that the recognition of a tRNA by an aminoacyl-tRNA synthetase does not seem to require the 3′ terminal part of the amino acid acceptor stem. As the importance of this part of the tRNA molecule during the aminoacylation process has been well established, it is possible that it is involved in determining the Vmax of the aminoacylation reaction, probably by positioning the 3′ terminal adenosine in the catalytic site of the enzyme. In conclusion, it appears that the specificity of the tRNA aminoacylation reaction proceeds through two discrimination mechanisms: the first one, measured by the Km, acts at the recognition level; the second one, which is more effective, is measured by the Vmax values. Competition phenomena have been observed between cognate and non-cognate tRNA species. They enhance the specificity of the tRNA aminoacylation, but their contribution to the specificity is low compared to that brought by Km and Vmax. Finally we found that a more rapid enzymatic deacylation of mischarged tRNA species (as compared to correctly charged ones) cannot be considered as a general mechanism for correction of misaminoacylation.

The Nucle(ol)ar Tif6p and Efl1p Are Required for a Late Cytoplasmic Step of Ribosome Synthesis

Deletion of elongation factor-like 1 (Efl1p), a cytoplasmic GTPase homologous to the ribosomal translocases EF-G/EF-2, results in nucle(ol)ar pre-rRNA processing and pre-60S subunits export defects. Efl1p interacts genetically with Tif6p, a nucle(ol)ar protein stably associated with pre-60S subunits and required for their synthesis and nuclear exit. In the absence of Efl1p, 50% of Tif6p is relocated to the cytoplasm. In vitro, the GTPase activity of Efl1p is stimulated by 60S, and Efl1p promotes the dissociation of Tif6p-60S complexes. We propose that Tif6p binds to the pre-60S subunits in the nucle(ol)us and escorts them to the cytoplasm where the GTPase activity of Efl1p triggers a late structural rearrangement, which facilitates the release of Tif6p and its recycling to the nucle(ol)us.

A Conserved Domain within Arc1p Delivers tRNA to Aminoacyl-tRNA Synthetases

Two yeast enzymes that catalyze aminoacylation of tRNAs, MetRS and GluRS, form a complex with the protein Arc1p. We show here that association of Arc1p with MetRS and GluRS is required in vivo for effective recruitment of the corresponding cognate tRNAs within this complex. Arc1p is linked to MetRS and GluRS through its amino-terminal domain, while its middle and carboxy-terminal parts comprise a novel tRNA-binding domain. This results in high affinity binding of cognate tRNAs and increased aminoacylation efficiency. These findings suggest that Arc1p operates as a mobile, trans-acting tRNA-binding synthetase domain and provide new insight into the role of eukaryotic multimeric synthetase complexes.

Purification et quelques proprietes de la phenylalanyl-tRNA synthetase de levure de boulangerie

Abstract Purification and some properties of phenylananyl-tRNA synthetase from bakers yeast Phenylalanyl-tRNA synthetase has been isolated from bakers yeast with a 600-fold purification. The different steps of the preparation are: (NH 4 ) 2 SO 4 precipitation of the 78 000 × g crude extract (between 50 and 65 % saturation), chromatography on DEAE-cellulose, CM-Sephadex C-50 and hydroxylapatite. The enzyme appears to be homogeneous on hydroxylapatite chromatography, sucrose gradient centrifugation and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation or equilibrium sedimentation studies give an average value of 220 000. Amino acid composition has been determined. No end group can be detected by the dansyl method. In the presence of either ATP and phenylalanine or tRNA Phe , the number of free thiol groups titrated with DTNB decreases. The enzyme is dissociated by 8 M urea or 1 % sodium dodecyl sulphate into two different equimolar components. The molecular weights of these 2 components were estimated to be 56 000 and 63 000, respectively, by polyacrylamide gel electrophoresis. The results suggest that the enzyme has a 4-subunits structure A 2 B 2 . The kinetics of the PP i -ATP exchange and aminoacylation reactions of tRNA Phe have been determined.

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 bakers 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.

Cloning, sequencing and characterization of the Saccharomyces cerevisiae URA7 gene encoding CTP synthetase

SummaryThe URA7 gene of Saccharomyces cerevisiae encodes CTP synthetase (EC 6.3.4.2) which catalyses the conversion of uridine 5′-triphosphate to cytidine 5′-triphosphate, the last step of the pyrimidine biosynthetic pathway. We have cloned and sequenced the URA 7 gene. The coding region is 1710 by long and the deduced protein sequence shows a strong degree of homology with bacterial and human CTP synthetases. Gene disruption shows that URA7 is not an essential gene: the level of the intracellular CTP pool is roughly the same in the deleted and the wild-type strains, suggesting that an alternative pathway for CTP synthesis exists in yeast. This could involve either a divergent duplicated gene or a different route beginning with the amination of uridine mono- or diphosphate.

Etude du complexe entre tRNAPhe et phenylalanyl-tRNA synthetase de levure

Abstract Study of the complex between tRNAPhe and phenylananyl-tRNA synthetase from yeast A stable and specific complex between the tRNAPhe and the phenylalanyl-tRNA synthetase from yeast has been isolated by sucrose gradient centrifugation. One molecule of tRNAPhe is combined with the tetrameric molecule of the enzyme. In the presence of tRNAPhe which is acylated by [14C]phenylalanine, a hydrolysis of the ester bond takes place during the isolation of the complex. This hydrolysis is strongly diminished in the absence of Mg2+. The interaction between tRNAPhe and the enzyme is strongly dependent upon the pH (optimum between 5.5 and 6.0) but is not influenced by either the increase in temperature (up to 37°) or the Mg2+. In the presence of the enzyme, the tRNAPhe is strongly protected against ribonuclease T1 action.

Identification of potential amino acid residues supporting anticodon recognition in yeast methionyl-tRNA synthetase.

Sequence comparisons among methionyl‐tRNA synthetases from different organisms reveal only one block of homology beyond the lasts β strand of the mononucleotide fold. We have introduced a series of semi‐conservative amino acid replacements in the conserved motif of yeast methionyl‐tRNA synthetase. The results indicate that replacements of two polar residues (Asn584 and Arg588) affected specifically the aminoacylation reaction. The location of these residues in the tertiary structure of the enzyme is compatible with a direct interaction of the amino acid side‐chains with the tRNA anticodon.

Yeast tRNAMet recognition by methionyl-tRNA synthetase requires determinants from the primary, secondary and tertiary structure: a review

The primordial role of the CAU anticodon in methionine identity of the tRNA has been established by others nearly a decade ago in Escherichia coli and yeast tRNA(Met). We show here that the CAU triplet alone is unable to confer methionine acceptance to a tRNA. This requires the contribution of the discriminatory base A73 and the non-anticodon bases of the anticodon loop. To better understand the functional communication between the anticodon and the active site, we analysed the binding and aminoacylation of tRNA(Met) based anticodon and acceptor-stem minihelices and of tRNA(Met) chimeras where the central core region of yeast tRNA(Met) is replaced by that of unusual mitochondrial forms lacking either a D-stem or a T-stem. These studies suggest that the high selectivity of the anticodon bases in tRNA(Met) implies the L-conformation of the tRNA and the presence of a D-stem. The importance of a L-structure for recognition of tRNA(Met) was also deduced from mutations of tertiary interactions known to play a general role in tRNA(Met) folding.