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Biomedical subjects

B Ehresmann

Publications and source records attributed to B Ehresmann.

162 records · Page 9Linked to original sources
PubMed1980

Identification of binding sites of turnip yellow mosaic virus protein and RNA by crosslinks induced in situ.

Turnip yellow mosaic virus RNA and protein could be crosslinked in situ by ultraviolet irradiation at pH 4.8 but not at pH 7.3, and by bisulphite treatment at pH 7.3. Crosslinked peptides could be located in the primary structure of the viral coat protein. Three regions were bound covalently by ultraviolet irradiation, and two of these three regions were bound also by bisulphite treatment. The yield of the crosslinking reaction could be high, indicating that almost all protein subunits of each virion reacted with the viral RNA. The crosslinked peptides contain most of the acidic and basic amino acids of the protein, often associated into pairs of opposite charge. Implications for the folding of the RNA into the virion and for models of RNA--protein interactions are discussed.

Cross-Linking Reagents↗
PubMed1980

Synthesis of a new reagent, ethyl 4-azidobenzoylaminoacetimidate, and its use for RNA-protein cross-linking within Escherichia coli ribosomal 30-S subunits.

A new reagent, ethyl 4-azidobenzoylaminoacetimidate, was prepared in a four-step synthesis starting from 4-aminobenzoic acid. This compound was used to cross-link RNA with proteins within the Escherichia coli 30-S ribosomal subunits. Following the reaction of the imidoester function with protein NH2 groups, photoactivation of the azide binds the other end of the reagent to RNA. The cross-linked proteins were labelled with 125I and identified by bidimensional gel electrophoresis. Proteins S3, S4, S5, S7, S9, S17, S18, and in a lower and more variable yield, S12, S13, S14 and S16 were bound to 16-S RNA. These results were confirmed by isolating cross-linked protein-oligonucleotide complexes from 30-S subunits containing 32P-labelled RNA.

Cross-Linking Reagents↗
PubMed1979

The complete nucleotide sequence of the ribosomal 16-S RNA from Excherichia coli. Experimental details and cistron heterogeneities.

The complete nucleotide sequence of the 16-S RNA from Escherichia coli has been determined using rapid RNA-sequencing gel methods. The experimental data are fully described in this paper. The specificities of the ribonucleases, especially the ribonuclease PhyI are discussed and the consequences of the persistence of stable secondary structure are considered. The proposed sequence contains 1541 nucleotides and agrees completely with the DNA sequence of the rrnB cistron deduced by Brosius, J., Palmer, M.L., Kennedy, P.J., and Noller, H.F. [Proc. Natl. Acad. Sci. U.S.A. (1978) 75, 4801-4805]. But there are several cistron heterogeneities of which we described 16 single-base heterogeneities, 7 of the deletion/insertion type and 9 of the transition or transversion type. Our observations suggest the existence, among the 7 ribosome RNA cistrons, of one or two mutated ones. The respective advantages and disadvantages of both RNA and DNA sequencing methods are discussed.

Base Sequence↗
PubMed1975

Effects of changes in growth rate on the levels of several aminoacyl-tRNA synthetases in yeast.

When the growth rate of yeast cells is decreased (for instance by transferring the cells from a rich medium to a poor one, or when the cells enter the stationary phase, or when growth is inhibited by cycloheximide, or when valine is removed from the medium supporting growth of a valine-requiring mutant, or when a thermosensitive mutant is shifted to the non-permissive temperature) there is a decrease in the levels of the four aminoacyl-tRNA synthetases tested. Conversely, an increase in the growth rate is accompanied by an increase in the levels of the four enzymes. But when the growth rate is slowed down by decreasing the temperature of the medium, no effect on the levels of the aminoacyl-tRNA synthetases is observed. These results are consistent with the concept of "metabolic regulation" proposed by Parker and neidhardt.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

Ribosomal 5S RNA from Xenopus laevis oocytes: conformation and interaction with transcription factor IIIA.

This review describes extensive studies on 5S rRNA from X laevis oocytes combining conformational analyses in solution (using a variety of chemical and enzymatic probes), computer modeling, site-directed mutagenesis, crosslinking and TFIIIA binding. The proposed 3-dimensional model adopts a Y-shaped structure with no tertiary interactions between the different domains of the RNA. The conserved nucleotides are not crucial for the tertiary folding but they maintain an intrinsic structure in the loop regions. The model was tested by the analysis of several 5S rRNA mutants. A series of 5S RNA mutants with defined block sequence changes in regions corresponding to each of the loop regions was constructed by in vitro transcription of the mutated genes. Our results show that none of the mutations perturbs the Y-shaped structure of the RNA, although they induce conformational changes restricted to the mutated regions. The interaction of the resulting 5S rRNA mutants with TFIIIA was determined by a direct binding assay. Only the mutations in the hinge region between the 3 helical domains have a significant effect on the binding for the protein. Finally, TFIIIA was crosslinked by the use of trans-diamminedichloroplatinum (II) to a region covering the fork region. Our results show that (i) the tertiary structure does not involve long-range interactions; (ii) the intrinsic structures in loops are strictly sequence-dependent; (iii) the hinge nucleotides govern the relative orientation of the 3 helical domains; (iv) TFIIIA recognizes essentially specific features of the tertiary structure of 5S rRNA.

Animals↗
PubMedDate not supplied

The relation between catalytic activity and gene regulation in the case of E coli threonyl-tRNA synthetase.

The expression of the gene for threonyl-tRNA synthetase (thrS) has previously been shown as being negatively autoregulated at the translational level. The region of the thrS leader mRNA responsible for that control is located immediately upstream of the ribosomal binding site, and was proposed to fold in a tRNA(Thr) anticodon arm-like structure. The present paper reviews experiments using enzymatic and chemical probes that prove the existence of a tRNA(Thr) anticodon-like structure in the thrS mRNA. These structural studies have also shown the presence of another arm upstream in the leader mRNA that has striking similarities with the acceptor arm of the tRNA(Thr) isoacceptors. This second arm was shown, by mutational analysis, to also be involved in thrS regulation. Footprinting experiments have shown that both the anticodon-like and the acceptor-like arms interact with the synthetase. Finally, the similarity of the interaction of the synthetase with its 2 RNA ligands (mRNA and tRNA) has been investigated by selecting and studying mutants of the synthetase itself. The observed correlation between regulatory and aminoacylation defects in these mutants strongly suggests that the synthetase recognizes similar regions of its 2 RNA ligands in an analogous manner.

Base Sequence↗
PubMedDate not supplied

Topography of the Escherichia coli ribosomal 30S subunit-initiation factor 2 complex.

The specific effect of the binding of initiation factor IF2 on E coli 16S rRNA within the [IF2/30S/GTP] complex has been probed by crosslinking experiment with trans-diamminedichloro platinum (II) and by phosphate alkylation with ethylnitrosourea. Several 16S rRNA fragments crosslinked to IF2 have been identified and are mostly located in the head and the lateral protrusion of the 30S subunit. The study of the effect of IF2 binding to the 30S subunit reveals that the factor does not tightly bind to the 16S rRNA and induces both isolated reductions and enhancements of phosphate reactivity in the 16S rRNA. Several of them are located near the binding site of IF2 and weak effects are observed in distant parts of the subunit. These results are discussed in the light of current knowledge of the topographical localization of IF2 with the 30S subunit and of its relation with function.

Base Sequence↗
PubMedDate not supplied

Synthesis and ribosome binding properties of model mRNAs modified with undecagold cluster.

The synthesis and purification of short model messenger RNAs modified with undecagold cluster are described. A monoamino undecagold cluster was introduced on the oxidized 3' cis-glycol group of the mRNA followed by reduction of the formed Schiff's base. The stability of the modified mRNA under the conditions used for in vitro messenger RNA translation is studied. The possibility of the formation of a specific translational initiation complex with bacterial ribosomes and modified mRNAs is shown. The results of these experiments indicate that the attachment of an undecagold cluster to a mRNA is a useful tool for electron microscopic and crystallographic studies.

Base Sequence↗
PubMedDate not supplied

A pseudoknot is required for efficient translational initiation and regulation of the Escherichia coli rpsO gene coding for ribosomal protein S15.

Escherichia coli ribosomal protein S15 down regulates its own synthesis by binding to its mRNA in a region overlapping the ribosome binding site, called the translational operator. This binding stabilizes a pseudoknot structure that exists in equilibrium with two stem-loop structures. When synthesized in excess over 16S rRNA, S15 binds to its translational operator and traps the ribosome on its loading site in a transient state, preventing the formation of the active ternary (30S-mRNA-rRNA(f)Met) complex. This inhibition can be suppressed by 16S rRNA, which displaces S15 from the mRNA. An extensive mutational analysis showed that the pseudoknot is the structural element required for S15 recognition and in vivo translational control. Specific sequence determinants are located in limited regions of the structure formed by the pseudoknot. An unexpected result is that the pseudoknot can exist in a variety of topologically equivalent structures recognizable and shapable by S15. Based on footprinting experiments and computer graphic modelling, S15 shields the two stems of the pseudoknot, sitting in the major groove of the coaxial stack.

Base Sequence↗