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B Ehresmann

Publications and source records attributed to B Ehresmann.

At least 145 records · Page 8Linked to original sources
PubMed1987

Probing the structure of RNAs in solution.

During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are oriented both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed.

Base Sequence↗
PubMed1987

Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA. A model for the interaction and the tertiary structure of the RNA binding site.

We have investigated in detail the secondary and tertiary structures of the 16 S rRNA binding site of protein S8 using a variety of chemical and enzymatic probes. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p- toluenesulfonate (at G(N-1) and U(N-3)) and with diethylpyrocarbonate (at A(N-7)). The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea. RNases T1, U2 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. The RNA region, encompassing nucleotides 582 to 656 was probed within: (1) the complete 16 S rRNA molecule; (2) a 16 S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S8-16 S rRNA complex; (4) the S8-RNA fragment complex; (5) the 30 S subunit. Cleavage or modification sites were detected by primer extension with reverse transcriptase. We present a three-dimensional model derived from mapping experiments and graphic modeling. Nucleotides in area 594-599/639-645 display unusual features: a non-canonical base-pair is formed between U598 and U641; and A595, A640 and A642 are bulging out of the major groove. The resulting helix is slightly unwound. Comparative analysis of probing experiments leads to several conclusions. (1) The synthesized fragment adopts the same conformation as the corresponding region in the complete RNA molecule, thus confirming the existence of independent folding domains in RNAs. (2) A long-range interaction involving cytosine 618 and its 5' phosphate occurs in 16 S rRNA but not in the fragment. (3) The fragment contains the complete information required for S8 binding. (4) The RNA binding site of S8 is centered in the major groove of the slightly unwound helix (594-599/639-645), with the three bulged adenines appearing as specific recognition sites. (5) This same region of the 16 S RNA is not exposed at the surface of the 30 S subunit.

Base Sequence↗
PubMed1987

trans-Diamminedichloroplatinum(II), a reversible RNA-protein cross-linking agent. Application to the ribosome and to an aminoacyl-tRNA synthetase/tRNA complex.

A new approach allowing detection of contact points between RNAs and proteins has been developed using trans-diamminedichloroplatinum(II) as the cross-linking reagent. The advantage of the method relies on the fact that the coordination bonds between platinum and the potential acceptors on proteins and nucleic acids (mainly S of cysteine or methionine residues; N of imidazole rings in histidine residues; N7 of guanine, N1 of adenine, and N3 of cytosine residues) can be reversed, so that the cross-linked oligonucleotides or peptides in contact within a complex can be analyzed directly. The method was worked out with the ribosome from Escherichia coli and the tRNAVal/valyl-tRNA synthetase system from the yeast Saccharomyces cerevisiae. In the first system the platinum approach permitted detection of ribosomal proteins cross-linked to 16S rRNA within the 30S subunits (mainly S18 and to a lower extent S3, S4, S11, and S13/S14); in the second system major oligonucleotides of tRNAVal cross-linked to valyl-tRNA synthetase were detected in the anticodon stem and loop, in the variable loop, and in the 3' terminal amino acid accepting region. These results are discussed in light of the current knowledge on ribosome and tRNAs and of potential applications of the methodology.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II). Characterization of two crosslinking sites in the tRNA.

Trans-diamminedichloroplatinum (II) was used to induce reversible crosslinks between EF-Tu and Phe-tRNA(Phe) within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex. Up to 40% of the complex was specifically converted into crosslinked species. Two crosslinking sites have been unambiguously identified. The major one encompassing nucleotides 58 to 65 is located in the 3'-part of the T-stem, and the minor one encompassing nucleotides 31 to 42 includes the anticodon loop and part of the 3'-strand of the anticodon stem.

Base Sequence↗
PubMed1987

Higher-order structure of domain III in Escherichia coli 16S ribosomal RNA, 30S subunit and 70S ribosome.

We have investigated in detail the conformation of domain III of 16S rRNA (nucleotides 913-1408), using a variety of chemical and enzymatic structure probes. The sites of reaction were identified by primer extension with reverse transcriptase using appropriate oligodeoxyribonucleotide primers. This study has been done on 16S rRNA in its naked form, in the 30S subunit and in the 70S ribosome. Data obtained with naked RNA broadly confirm the secondary structure model proposed essentially by comparative sequence analysis, and allow identification of nucleotides involved in tertiary interactions. Our results are in reasonably good agreement with structure probing experiments of Moazed et al. [1]. However, several discrepancies have been observed. Within the 30S subunit, a high number of nucleotides become unreactive whereas other nucleotides show an enhanced reactivity. This probably reflects local conformational changes. Interestingly, they are located in strategic regions of the RNA, e.g. around C1400 (involved in tRNA binding) and C1192 (involved in spectinomycin recognition). Results are also discussed together with the topographical localization of the ribosomal proteins in this area. The study on the 70S particle allows identification of regions at the interface of subunits or exposed at the surface of the ribosome.

Base Sequence↗
PubMed1987

Importance of conserved residues for the conformation of the T-loop in tRNAs.

The conformation of the T-loop of yeast tRNA(Asp) was studied by structural mapping techniques using chemical and enzymatic probes and by three-dimensional graphics modeling with the known crystallographic structures of tRNAs as references. The structural importance of C61 (conserved in the T-stem of all tRNAs) for the loop conformation was directly checked by ethylnitrosourea phosphate alkylation, either on the 3'-half tRNAAsp molecule or on a variant in which C61 was replaced by U61. The reactivity of P60 against ethylnitrosourea alkylation in the variant emphasizes the role of the hydrogen bond between this phosphate and position N4 of C61 for stabilizing the conformation of the T-loop. Experiments on several tRNA variants, containing C61 but altered in the sequence or in the length of the T-loop, indicate that other structural features help to stabilize the hydrogen bond network around P60. Evidence is presented that the reverse Hoogsteen base pair T54-A58 contributes to this stabilization by maintaining the hydrogen bonding between the 2'OH of ribose 58 and P60. Using graphics modeling and based on the chemical data. T-loops of several variants were constructed. It appears that both the constant length of the T-loop and the presence of psi 55 are crucial for the correct interaction between the T- and D-loops. The conclusion of this study is that the T-loop in tRNA possesses an intrinsic conformation (mainly governed by the constant residues) existing primarily without the structural context of the entire tRNA molecule.

Base Composition↗
PubMed1986

Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3.

The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far.

Base Sequence↗
PubMed1986

Binding of Escherichia coli ribosomal protein S8 to 16S rRNA: kinetic and thermodynamic characterization.

A sensitive membrane filter assay has been used to examine the kinetic and equilibrium properties of the interactions between Escherichia coli ribosomal protein S8 and 16S rRNA. In standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 0.35 M KCl) the apparent association constant is 5 +/- 0.5 X 10(-7) M-1. The interaction is highly specific, and the kinetics of the reaction are consistent with the apparent association constant. Nevertheless, the rate of association is somewhat slower than that expected for a diffusion-controlled reaction, suggesting some steric constraint. The association is only slightly affected by temperature (delta H = -1.8 kcal/mol). The entropy change [delta S = +29 cal/(mol K)] is clearly the main driving force for the reaction. The salt dependence of Ka reveals that five ions are released upon binding at pH 7.5 and in the presence of 10 mM magnesium. The substitution of various anions for Cl- has an appreciable effect on the magnitude of Ka, following the order CH3COO- greater than Cl- greater than Br-, thus indicating the existence of anion binding site(s) on S8. An equal number of ions were released when Cl- was replaced by CH3COO-, but the absence of anion release upon binding cannot be excluded. On the other hand, the free energy of binding appears not to be exclusively electrostatic in nature. The effect of pH on both temperature and ionic strength dependence of Ka has been examined. It appears that protonation of residue(s) (with pK congruent to 9) increases the affinity via a generalized charge effect. On the other hand, deprotonation of some residue(s) with a pK congruent to 5-6 seems to be required for binding. Furthermore, the unique cysteine present in S8 was shown to be essential for binding.

Escherichia coli↗
PubMed1985

Conformation of yeast 18S rRNA. Direct chemical probing of the 5' domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible.

The structure of the 5' domain of yeast 18S rRNA has been probed by dimethyl sulfate (DMS), either in "native" deproteinized molecules or in the 40S ribosomal subunits. DMS-reacted RNA has been used as a template for reverse transcription and a large number of reactive sites, corresponding to all types of bases have been mapped by a primer extension procedure, taking advantage of blocks in cDNA elongation immediately upstream from bases methylated at atom positions involved in the base-pair recognition of the template. Since the same atom positions are protected from DMS in base-paired nucleotides, the secondary structure status of each nucleotide can be directly assessed in this procedure, thus allowing to evaluate the potential contribution of proteins in modulating subunit rRNA conformation. While the DMS probing of deproteinized rRNA confirms a number of helical stems predicted by phylogenetic comparisons, it is remarkable that a few additional base-pairings, while proven by the comparative analysis, appear to require the presence of the bound ribosomal subunit proteins to be stabilized.

Hydrogen Bonding↗
PubMed1984

Chemical conversion of cytidine residues into 4-thiouridines in yeast tRNAPhe. Determination of the modified cytidines.

Treatment of yeast phenylalanine tRNA with pressurized hydrogen sulfide results in conversion of cytidine residues into 4-thiouridine residues. Under conditions leading to an average modification of one cytidine per tRNA molecule 9 positions are thiolated. The 4-thiouridine residues are distributed along the tRNA molecule. Four of the reactive cytidines are located in single-stranded regions: Cm32 , C60 , C74 and C75 . The five others are located in base pairs: C2, C27, C56 , C61 and C63 . Importance of replacement of an amino group by a thiol group on hydrogen bonding and on biological activity of the modified tRNA is discussed.

Autoradiography↗
PubMed1984

Cross-linking of the anticodon of Escherichia coli and Bacillus subtilis acetylvalyl-tRNA to the ribosomal P site. Characterization of a unique site in both E. coli 16S and yeast 18S ribosomal RNA.

The nucleotide residues involved in the cross-link between P site bound acetylvalyl-tRNA (AcVal-tRNA) and 16-18S rRNA have been identified. This cross-link was formed by irradiation of Escherichia coli or Bacillus subtilis AcVal-tRNA bound to the P site of E. coli ribosomes or by irradiation of E. coli AcVal-tRNA bound to the P site of yeast ribosomes. The three cross-linked RNA heterodimers were obtained in 10-35% purity by disruption of the irradiated ribosome-tRNA complex with sodium dodecyl sulfate followed by sucrose gradient centrifugation. After total digestion with RNase T1, and labeling at either the 5'- or the 3'-end, the cross-linked oligomers could be identified and isolated before and after photolytic splitting of the cross-link. One of the oligomers was shown to be UACACACCG, a unique rRNA nonamer present in an evolutionarily conserved region. This oligomer was found in all three heterodimers. The other oligomer of the dimer had the sequence expected for the RNase T1 product encompassing the anticodon of the tRNA used. The precise site of cross-linking was determined by two novel methods. Bisulfite modification of the oligonucleotide dimer converted all C residues to U, except for any cross-linked C which would be resistant by being part of a cyclobutane dimer. Sequencing gel analysis of the UACACACCG oligomer showed that the C residue protected was the 3'-penultimate C residue, C1400 in E. coli rRNA or C1626 in yeast rRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticodon↗
PubMed1983

Nucleotides in 16S rRNA that are required in unmodified form for features recognized by ribosomal protein S8.

Nucleotides in 16S rRNA which are required in unmodified form for specific recognition of ribosomal protein S8 from Escherichia coli were identified using a damage-selection experimental approach. Prior to complex formation with S8, 16S rRNA was treated under fully denaturing conditions with either diethyl pyrocarbonate or 25% hydrazine. Following separation of bound from unbound fragments of RNA, those associated with S8 were analyzed for their content of modified bases by treatment with aniline. Nucleotides found to be consistently unmodified in such fragments were located near the base of a stable helix (encompassing bases 581-656) or near the apex of the helix on the 3' proximal side. A minor S8 ribonucleoprotein particle was found to contain fragments which extended in the 3' direction to position 671.

Base Sequence↗
PubMed1983

Effect of elongation factor Tu on the conformation of phenylalanyl-tRNAPhe.

Structural features of the tRNAPhe molecule upon ternary complex formation with the bacterial elongation factor Tu were investigated. Phosphodiester bonds at positions 18 and 34 were found to be labilized in bound tRNA. Conversely, a higher stability of the phosphodiester links at positions 20, 21 and 36 was detected. Using ethylnitrosourea as a chemical probe a conformational change occurring at phosphate position 53 was observed in complexed tRNA. These results are interpreted by a structural rearrangement of the nucleic acid induced by complex formation.

Chemical Phenomena↗
PubMed1982

Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide.

Bacterial Proteins↗
PubMed1982

Crosslinking of N-acetyl-phenylalanyl [s4U]tRNAPhe to protein S10 in the ribosomal P site.

In order to identify ribosomal components involved in the peptidyl-tRNA binding site on the ribosome, tRNAPhe molecules were prepared in which cytidine residues had been chemically converted into 4-thiouridine (S4U). This nucleoside is photoactive at 335 nm and able to form covalent bonds with nearby nucleophilic groups. The thiolated AcPhe-tRNAPhe was bound to the ribosomal P site in the presence of poly(U) as verified by puromycin reactivity. Direct irradiation of the AcPhe-[s4U]tRNAPhe poly(U) 70-S ribosome complex induced crosslinking of the tRNA molecule exclusively to 30-S subunits. Analysis of the covalent complex revealed that AcPhe-[s4U]tRNAPhe was specifically crosslinked to protein S10.

Base Sequence↗
PubMed1981

Identification of a 16-S RNA fragment crosslinked to protein S1 within Escherichia coli ribosomal 30-S subunits by the use of a crosslinking reagent: ethyl 4-azidobenzoylaminoacetimidate.

The bifunctional reagent ethyl 4-azidobenzoylaminoacetimidate was used to crosslink specifically ribosomal protein S1 to 16-S RNA within 30-S subunits. The reagent was attached to isolated protein S1. The modified protein was reassociated with protein-S1-depleted 30-S subunits and then crosslinked to the RNA molecule. The covalently bound 16-S RNA-protein S1 complex was isolated and the RNA fragment C-U-A-A-C-G-C-G-U-U-A-A-G-U-C-G-A-C-C-G-C-C-U-G-G-G-G-A-G (positions 861-889) was characterized to be crosslinked to protein S1.

Base Sequence↗
PubMed1980

Effect of ultraviolet irradiation on 30-S ribosomal subunits. Identification of the RNA region crosslinked to protein S7.

The effects of ultraviolet irradiation on Escherichia coli 30-S ribosomal subunits were studied. At the doses of radiation used in this work (0-4.5 x 10(5) quanta/30-S subunit), only protein S7 was found to be significantly crosslinked to the 16-S RNA. In conditions where 25% of the protein was covalently crosslinked, the ability of the irradiated 30-S subunits to reassociate with 50-S subunits and their activity in polyphenylalanine synthesis decreased strongly. Similar results were obtained by irradiation with a germicide lamp (254 nm) or with a monochromatic ultraviolet light at 248 nm. No additional proteins were crosslinked to the 16-S RNA by irradiating 30-S subunits depleted in protein S1 or 70-S ribosomes. The covalent complex of 16-S RNA and protein S7 was isolated and digested by T1 ribonuclease. The oligonucleotide remaining attached to the crosslinked protein was characterised as A-C-C-U-C-G [position 1261 - 1266, see the sequence published by Carbon et al. (1979) Eur. J. Biochem. 160, 399-410]. Analysis of this fragment suggests that protein S7 was linked to the cytosine at position 1265 in the RNA sequence.

Dose-Response Relationship, Radiation↗