Biomedical subjects

J P Ebel

Publications and source records attributed to J P Ebel.

At least 19 recordsLinked to original sources

PubMed1993

An in vitro/in vivo correlation for the disintegration and onset of drug release from enteric-coated pellets.

An empirical mass-transfer model for enteric-coating dissolution that uses in vivo dissolution data to characterize the pH-dependent solubility properties of the polymer film and a mass-transfer coefficient determined from in vivo dissolution or disintegration studies is developed. Once the in vivo mass-transfer coefficient has been evaluated, it can be used in conjunction with in vitro dissolution data from other formulations to predict the in vivo time to disintegration and onset of drug release. Results of in vitro dissolution experiments using the USP basket dissolution apparatus and in vivo disintegration experiments using gamma scintigraphy with four enteric-coated pellet formulations are presented. The good agreement among the in vivo mass-transfer coefficients that were determined supports the validity of the model.

Adult↗

PubMed1991

Evolution of aminoacyl-tRNA synthetase quaternary structure and activity: Saccharomyces cerevisiae mitochondrial phenylalanyl-tRNA synthetase.

Phenylalanyl-tRNA synthetases [L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20] from Escherichia coli, yeast cytoplasm, and mammalian cytoplasm have an unusual conserved alpha 2 beta 2 quaternary structure that is shared by only one other aminoacyl-tRNA synthetase. Both subunits are required for activity. We show here that a single mitochondrial polypeptide from Saccharomyces cerevisiae is an active phenylalanyl-tRNA synthetase. This protein (the MSF1 gene product) is active as a monomer. It has all three characteristic sequence motifs of the class II aminoacyl-tRNA synthetases, and its activity may result from the recruitment of additional sequences into an alpha-subunit-like structure.

Amino Acid Sequence↗

PubMed1991

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes.

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNAPhe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNAPhe/(U)9/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319-322).

Crystallography↗

PubMed1991

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 last beta 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.

Amino Acid Sequence↗

PubMed1991

Interaction of the tRNA(Phe) acceptor end with the synthetase involves a sequence common to yeast and Escherichia coli phenylalanyl-tRNA synthetases.

Modified lysines resulting from the cross-linking of the 3' end of tRNA(Phe) to yeast phenylalanyl-tRNA synthetase (an enzyme with an alpha 2 beta 2 structure) have been characterized by sequencing the labeled chymotryptic peptides that were isolated by means of gel filtration and reversed-phase chromatography. The analysis showed that Lys131 and Lys436 in the alpha subunit are the target sites of periodate-oxidized tRNA(Phe). Mutant protein with a Lys----Asn substitution established that each lysine contributes to the binding of the tRNA but is not essential for catalysis. The major labeled lysine (K131) belongs to the sequence IALQDKL (residues 126-132), which shares three identities with the peptide sequence ADKL found around the tRNAox-labeled Lys61 in the large subunit of Escherichia coli phenylalanyl-tRNA synthetase [Hountondji, C., Schmitter, J. M., Beauvallet, C., & Blanquet, S. (1987) Biochemistry 26, 5433-5439].

Amino Acid Sequence↗

PubMed1991

Analysis of mutant tRNA gene transcripts in vivo in Saccharomyces cerevisiae by abortive primer extension.

When the primer extension of a synthetic oligonucleotide hybridized to a complementary region of RNA is made in the presence of only three deoxyribonucleosides triphosphates, elongation of the primer stops as soon as the missing nucleotide is needed. This abortive primer extension assay has been adapted to analyse tRNA gene transcripts and has two main advantages. First it is specific and allows the identification of particular tRNA gene products in an homologous system provided the gene bears a point mutation. Second, it is highly sensitive and can be used to complement and confirm results of Northern blot hybridization. This assay should be a useful tool in the further in vivo study of the transcription and processing of particular tRNA genes in the homologous system. In this report the expression of wild-type and mutant yeast Sup4- tyrosine inserting suppressor gene was studied.

Base Sequence↗

PubMed1990

Escherichia coli threonyl-tRNA synthetase and tRNA(Thr) modulate the binding of the ribosome to the translational initiation site of the thrS mRNA.

Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm. This binding is restricted to the stem-loop structure of the second site. However, the interaction of the enzyme with this site fails to occlude ribosome binding. tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex. Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase. This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell.

Anticodon↗

PubMed1990

Binding of initiation factor 2 and initiator tRNA to the Escherichia coli 30S ribosomal subunit induces allosteric transitions in 16S rRNA.

The specific effect of the binding of IF2 and initiator fMET-tRNA(fMet) on Escherichia coli 16S rRNA has been probed by phosphate alkylation with ethylnitrosourea. The results show that IF2 does not significantly shield portions of 16S rRNA but induces both reductions and enhancements of reactivity scattered in the entire molecule. Most of them are topographically constrained in a region corresponding to the cleft, the lateral protrusion, and the part of the head facing the protrusion (positions 694, 771, 791, 1225, 1268, 1398, 1401, 1504, and 1527). Weak effects are also observed in distant parts of the subunit (positions 301, 302, 492, and 1428). All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG as messenger. The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding. Most of the changes correspond to reduced reactivities (positions 791, 1222, 1263, 1393, 1395, 1430, 1431, 1504, 1528, and 1529), while enhanced reactivities are observed at positions 708, 709, and 1398. Functional implications are discussed, which stress the dynamic properties of the ribosome.

Allosteric Regulation↗

PubMed1990

The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex.

The conformation of the E. coli initiator tRNA and of the 16S rRNA at different steps leading to the 30S.IF2.fMet-ARN(fMet).AUG.GTP complex has been investigated using several structure-specific probes. As compared to elongator tRNA, the initiator tRNA exhibits specific structural features in the anticodon arm, the T and D loops and the acceptor arm. Initiation factor 2 (IF2) interacts with the T-loop and the minor groove of the T stem of the RNA, and induces an increased flexibility in the anticodon arm. In the 30S initiation complex, additional protection is observed in the acceptor stem and in the anticodon arm of the tRNA. Within the 30S subunit, IF2 does not significantly shield defined portions of 16S rRNA, but induces both reduction and enhancement of reactivity scattered in the entire molecule. Most are constrained in a region corresponding to the cleft, the lateral protrusion and the part of the head facing the protrusion. All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG message. The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding.

Base Sequence↗

PubMed1990

Search of essential parameters for the aminoacylation of viral tRNA-like molecules. Comparison with canonical transfer RNAs.

Comparative structural and functional results on the valine and tyrosine accepting tRNA-like molecules from turnip yellow mosaic virus (TYMV) and brome mosaic virus (BMV), and the corresponding cognate yeast tRNAs are presented. Novel experiments on TYMV RNA include design of variant genes of the tRNA-like domain and their transcription in vitro by T7 RNA polymerase, analysis of their valylation catalyzed by yeast valyl-tRNA synthetase, and structural mapping with dimethyl sulfate and carbodiimide combined with graphical modelling. Particular emphasis is given to conformational effects affecting the valylation capacity of the TYMV tRNA-like molecule (e.g., the effect of the U43----C43 mutation). The contacts of the TYMV and BMV RNAs with valyl- and tyrosyl-tRNA synthetases are compared with the positions in the molecules affecting their aminoacylation capacities. Finally, the involvement of the putative valine and tyrosine anticodons in the tRNA-like valylation and tyrosylation reactions is discussed. While an anticodon-like sequence participates in the valine identity of TYMV RNA, this seems not to be the case for the tyrosine identity of BMV RNA despite the fact that the tyrosine anticodon has been shown to be involved in the tyrosylation of canonical tRNA.

Anticodon↗

PubMed1990

Translational control of ribosomal protein S15.

The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

Base Sequence↗

PubMed1990

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome.

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.

Anticodon↗

PubMed1990

Solution structure of human U1 snRNA. Derivation of a possible three-dimensional model.

The solution structure of human U1 snRNA was investigated by using base-specific chemical probes (dimethylsulfate, carbodiimide, diethylpyrocarbonate) and RNase V1. Chemical reagents were employed under various conditions of salt and temperature and allowed information at the Watson-Crick base-pairing positions to be obtained for 66% of the U1 snRNA bases. Double-stranded or stacked regions were examined with RNase V1. The dat gained from these experiments extend and support the previous 2D model for U1snRNA. However, to elucidate some aspects of the solution data that could not be accounted for by the secondary structure model, the information gathered from structure probing was used to provide the experimental basis required to construct and to test a tertiary structure model by computer graphics modeling. As a result, U1 snRNA is shown to adopt an asymmetrical X-shape that is formed by two helical domains, each one being generated by coaxial stacking of helices at the U1 snRNA cruciform. Chemical reactivities and model building show that a few nucleotides, previously proposed to be unpaired, can form A.G and U.U non Watson-Crick base-pairs, notably in stem-loop B. The structural model we propose for regions G12 to A124 integrates stereochemical constraints and is based both on solution structure data and sequence comparisons between U1 snRNAs.

Alkylating Agents↗

PubMed1990

Construction of a FRS1-FRS2 operon encoding the structural genes for the alpha and beta subunits of yeast phenylalanyl-tRNA synthetase and its use in deletion analysis.

FRS1 and FRS2, the structural genes encoding the large (alpha) and small (beta) subunits of yeast phenylalanyl-tRNA synthetase (PheRS) were placed under the control of the lacZ promoter by creating an artificial operon. The FRS2 gene was fused next to the promoter, followed by a 14 base pair intergenic sequence containing a translation reinitiation site in front of the FRS1 coding sequences. The engineered PheRS has 16 N-terminal amino acids from beta-galactosidase fused to the beta subunit. However, the purified protein shows a Km value for tRNA(Phe) that is indistinguishable from that of the the native enzyme. The product of the FRS2-FRS1 operon is not able to complement thermosensitive E. coli PheRS, indicating the lack of heterologous aminoacylation in vivo. We made a deletion in the FRS2 gene that removed about 150 amino terminal residues of the beta subunit. The truncated protein showed intact ATP-PPi exchange, whereas tRNA aminoacylation was lost. This result is similar to that of limited proteolysis performed on the native enzyme that yielded a tetrameric alpha 2 beta'2 structure, able to form aminoacyladenylate but unable to bind tRNA(Phe). A deletion of 50 amino acids from the carboxyl terminus of the beta chain resulted in the loss of both enzyme activities; this suggests the participation of the C-terminal end of the beta subunit in the active site or in subunit assembly to yield a tetrameric functional enzyme.

Acylation↗

PubMed1990

Relaxation of a transfer RNA specificity by removal of modified nucleotides.

The molecular recognition of specific transfer RNAs by the appropriate aminoacyl-tRNA synthetase is an important step in determining the accuracy of translation of the genetic message from nucleic acids into proteins. Recent studies using variant tRNAs with specific sequence modifications have indicated particular regions that determine their identity. Here we consider whether the base modifications commonly found in tRNAs contribute to their identity. Although unmodified tRNA(Asp) is charged with aspartate as efficiently as the modified native tRNA, it is mischarged with arginine with considerably increased efficiency. Our results indicate that post-transcriptional modification of tRNAs introduces structural 'anti-determinants', restricting the efficiency with which the tRNAs are charged with inappropriate amino acids.

Arginine↗

PubMed1990

Target site of Escherichia coli ribosomal protein S15 on its messenger RNA. Conformation and interaction with the protein.

The regulatory site of ribosomal protein S15 has been located in the 5' non-coding region of the messenger, overlapping with the ribosome loading site. The conformation of an in vitro synthesized mRNA fragment, covering the 105 nucleotides upstream from the initiation codon and the four first codons of protein S15, has been monitored using chemical probes and RNase V1. Our results show that the RNA is organized into three domains. Domains I and II, located in the 5' part of the mRNA transcript, are folded into stable stem-loop structures. The 3'-terminal domain (III), which contains the Shine-Dalgarno sequence and the AUG initiation codon, appears to adopt alternative conformations. One of them corresponds to a rather unstable stem-loop structure in which the Shine-Dalgarno sequence is paired. An alternative potential structure involves a "pseudo-knot" interaction between bases of this domain and bases in the loop of domain II. The conformation of several RNA variants has also been investigated. The deletion of the 5'-proximal stem-loop structure (domain I), which has no effect on the regulation, does not perturb the conformation of the two other domains. The deletion of domain II, leading to a loss of regulatory control, prevents the formation of the potential helix involved in the pseudo-knot structure and results in a stabilization of the alternative stem-loop structure in domain III. The replacement of another base in domain III involved in pairing in the two alternative structures mentioned above should induce a destabilization of both structures and results in a loss of the translational control. However, the replacement of another base in domain III, which does not abolish the control, results in the loss of the conformational heterogeneity in this domain and yields a stable conformation corresponding to the pseudo-knot structure. Thus, it appears that any mutation that disrupts or alters the formation of the pseudo-knot impairs the regulatory mechanism. Footprinting experiments show that protein S15 is able to bind to the synthesized fragment and provide evidence that the protein triggers the formation of the pseudo-knot conformation. A mechanism can be postulated in which the regulatory protein stabilizes this particular structure, thus impeding ribosome initiation.

Base Composition↗

PubMed1990

Repair of acetyl-aminofluorene modified pBR322 DNA in Xenopus laevis oocytes and eggs; effect of diadenosine tetraphosphate.

Using Xenopus laevis oocytes and unfertilized eggs, we have developed a system which allows the study of DNA repair upon microinjection of pBR 322 DNA which has been previously modified in vitro by N-acetyl-aminofluorene, under controlled conditions. In unfertilized eggs, an efficient repair of pBR-18AAF DNA takes place, leading to a restoration of the transforming activity of the plasmid DNA towards Escherichia coli. The repaired DNA is even efficiently replicated, the egg being "activated" by the microinjection. In the oocyte, a partial repair is observed as shown by the incorporation of labelled dCTP in the modified plasmid DNA, even in the presence of aphidicolin, an inhibitor of DNA polymerase alpha. However, the repair appears to be very limited, since it does not restore the transforming activity of the modified plasmid DNA. This inefficient repair in the oocyte may be due to the rapid packaging of foreign DNA into a minichromosome and/or to a very low level of DNA polymerase beta. This system was used to study the effect of diadenosine tetraphosphate (Ap4A) on DNA repair. Ap4A seems not to interfere with repair processes in the oocyte, but significantly inhibits the replication following the repair of AAF-modified plasmid DNA in unfertilized eggs. These results suggest that Ap4A could be involved in switching off the replication machinery when DNA is badly damaged, thus helping to avoid the perpetuation of DNA modifications in the daughter cells. This hypothesis is consistent with many previous reports on the accumulation of dinucleoside polyphosphates under stress conditions, which are known to result in modification of DNA.

2-Acetylaminofluorene↗

PubMed1990

Yeast methionyl-tRNA synthetase: analysis of the N-terminal extension and the putative tRNA anticodon binding region by site-directed mutagenesis.

Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. 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 amino-acylation steps 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 3-D 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 of the catalytic residues. The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.

Adenosine Triphosphate↗