Biomedical subjects

I Weygand-Durasevic

Publications and source records attributed to I Weygand-Durasevic.

10 recordsLinked to original sources

PubMed2000

Characterization of yeast seryl-tRNA synthetase active site mutants with improved discrimination against substrate analogues.

The involvement of amino acids within the motif 2 loop of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) in serine and ATP binding was demonstrated previously [B. Lenhard et al., J. Biol. Chem. 272 (1997) 1136-1141]. In our attempt to analyze the structural basis for the substrate specificity and to explore further the catalytic mechanism employed by S. cerevisiae SerRS, two new active site mutants, SerRS11 and SerRS12, were constructed. The catalytic effects of amino acid replacement at positions Lys287, Asp288 and Ala289 with purified wild-type and mutant seryl-tRNA synthetases were tested. The alteration of these semi-conserved amino acids interferes with tRNA-dependent optimization of serine recognition. Additionally, mutated enzymes SerRS11 (Lys287Thr, Asp288Tyr, Ala289Val) and SerRS12 (Lys287Arg) are less sensitive to inhibition by two competitive inhibitors: serine hydroxamate, an analogue of serine, and 5'-O-[N-(L-seryl)-sulfamoyl]adenosine, a stable analogue of aminoacyl adenylate, than the wild-type enzyme. SerRS mutants also display different activation kinetics for serine and serine hydroxamate, indicating that specificity toward the substrates is modulated by amino acid replacement in the motif 2 loop.

Kinetics↗

PubMed1998

C-terminal truncation of yeast SerRS is toxic for Saccharomyces cerevisiae due to altered mechanism of substrate recognition.

Like all other eukaryal cytosolic seryl-tRNA synthetase (SerRS) enzymes, Saccharomyces cerevisiae SerRS contains a C-terminal extension not found in the enzymes of eubacterial and archaeal origin. Overexpression of C-terminally truncated SerRS lacking the 20-amino acid appended domain (SerRSC20) is toxic to S. cerevisiae possibly because of altered substrate recognition. Compared to wild-type SerRS the truncated enzyme displays impaired tRNA-dependent serine recognition and is less stable. This suggests that the C-terminal peptide is important for the formation or maintenance of the enzyme structure optimal for substrate binding and catalysis.

Amino Acid Sequence↗

PubMed1997

Matrix-assisted laser desorption/ionisation mass spectrometry of transfer ribonucleic acids isolated from yeast.

tRNATyr and tRNASer purified from bulk brewer's yeast tRNA were subjected to analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Choosing a mixture of 2,4,6- and 2,3,4-trihydroxy-acetophenone and diammonium citrate as matrix a mass resolution of up to 220 (FWHM) was achieved in the linear mode of operation. Cation adduct suppression by addition of cation exchange beads and a chelating agent (CDTA) is shown to substantially improve mass resolution for this class of molecules.

RNA, Fungal↗

PubMed1996

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis.

Adenosine Triphosphate↗

PubMed1995

Substrate selection by aminoacyl-tRNA synthetases.

The integration of genetic and biochemical approaches to study the crystal structure of the glutaminyl-tRNA synthetase (GlnRS):tRNA(Gln):ATP complex has elucidated the mechanism by which GlnRS selects its cognate tRNA for aminoacylation. Three principal types of interaction have been identified: interaction with specific bases in the cognate tRNA, rejection of non-cognate tRNAs, and activation of the active site upon cognate tRNA binding. The recent solving of the crystal structure of tryptophanyl-tRNA synthetase (TrpRS) has allowed comparable studies to be initiated in an aminoacyl-tRNA synthetase which, unlike GlnRS, does not require tRNA binding prior to amino acid activation.

Amino Acyl-tRNA Synthetases↗

PubMed1994

Thiobacillus ferrooxidans tyrosyl-tRNA synthetase functions in vivo in Escherichia coli.

The tyrosyl-tRNA synthetase gene (tyrZ) from Thiobacillus ferrooxidans, an acidophilic, autotrophic, gram-negative bacterium that participates in bioleaching of minerals, was cloned and sequenced. The encoded polypeptide (TyrRZ) is 407 amino acids in length (molecular mass; 38 kDa). The predicted protein sequence has an extensive overall identity (44%) to the sequence of the protein encoded by the Bacillus subtilus tyrZ gene, one of the two genes encoding tyrosyl-tRNA synthetases in this microorganism. Alignment with Escherichia coli TyrRS revealed limited overall identity (24%), except in the regions of the signature sequence for class I aminoacyl-tRNA synthetases. Complementation of an E. coli strain with a thermosensitive mutation in TyrRS showed that the protein encoded by the T. ferrooxidans tyrZ gene is functional and recognizes the E. coli tRNA(Tyr) as a substrate. TyrZ is a single-copy gene as revealed by Southern blot analysis. The gene was localized upstream from the putative promoters of the rrnT2 ribosomal RNA operon. Although no rho-independent transcription terminator was found between the two genes, a 1.3-kb RNA hybridized to a DNA probe derived from the tyrZ gene. The functional relationship between these two transcription units is discussed.

Acidithiobacillus thiooxidans↗

PubMed1990

Sequence-specific spin labeling of DNA.

Several DNA fragments deriving from plasmid pBR322 were used to determine the modification sites caused by the reaction with alkylating spin-labeling probes. At a high spin-label concentration, all guanines became alkylated, causing the cleavage of the phosphodiester bonds upon the treatment with piperidine. The lengths of the breakage products of 5'-end labeled DNA treated with spin labels were compared with the length of DNA scission products generated by Maxam-Gilbert procedure for DNA sequence analysis. The distribution of the guanine modifications is dependent on the amount of the reagent used for the alkylation and the ionic conditions of the reaction. The frequency of alkylation by spin labels was greatly enhanced within continuous runs of guanines in DNA. The stabilization of the DNA structure by magnesium or spermine directs the spin-label binding specifically to the most exposed region of DNA fragment containing GGTGG sequence. The sequence-dependent interaction of spin labels with DNA enables the development of the method for the selective spin labeling of DNA molecule.

Alkylation↗

PubMed1989

Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme.

MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA aminoacylation step than on the adenylate formation step. The Km for ATP and methionine are unaltered indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3 which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the three-dimension structure of the homologous E. coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide-binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration to the catalytic residues.

Amino Acid Sequence↗

PubMed1987

Cloning and characterization of the gene coding for cytoplasmic seryl-tRNA synthetase from Saccharomyces cerevisiae.

We have screened a Saccharomyces cerevisiae expression library with antibodies against seryl-tRNA synthetase (SerRS) from baker's yeast. In this way we obtained clones which contain serS, the structural gene for seryl-tRNA synthetase. Genomic Southern blots show that the serS gene resides on a 5.0 kb SalI fragment. Nucleotide sequence analysis of the genes revealed a single open reading frame from which we deduced the amino acid sequence of the enzyme consistent with that of two peptides isolated from SerRS. The enzyme is comprised of 462 amino acids consistent with earlier determinations of its molecular weight. The codon usage of serS is typical of abundant yeast proteins. Nuclease S1 analysis of serS mRNA defined the RNA initiation site 20-40 bases downstream from an AT rich sequence containing the TATA box and 21-39 nucleotides upstream of the translation initiation codon. Yeast strains transformed with the cloned gene overproduce seryl-tRNA synthetase in vivo.

Amino Acid Sequence↗

PubMed1981

The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr.

The interactions of yeast tRNATyr, spin-labelled at position i6A-37 next to the anticodon, with EF-Tu . GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied. The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion. Upon anticodon-anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed.

Anticodon↗