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C Ehresmann

Publications and source records attributed to C Ehresmann.

At least 145 records · Page 8Linked to original sources
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

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↗
PubMed1984

Two-dimensional gel electrophoresis technique for determination of the cross-linked nucleotides in cleavable covalent RNA-RNA complexes. Application to Escherichia coli and Bacillus subtilis acetylvalyl-tRNA covalently linked to E. coli 16S and yeast 18S ribosomal RNA.

We have developed a new method which yields in a single step the site of cross-linking between two oligonucleotides covalently linked by a cleavable bond. The isolated duplex, labeled at both 5'-ends, is split randomly and then analyzed by diagonal gel electrophoresis with cleavage of the cross-link between the two gel dimensions. Digestion products which do not contain the cross-link migrate along the diagonal, while products resulting from cleavage of the cross-link migrate as off-diagonal products. The site of cross-linking is determined by analysis of both diagonal and off-diagonal products. This method was successfully applied to three different oligonucleotide duplexes isolated by T1 RNase digestion from Escherichia coli tRNA covalently linked at the ribosomal P site to either Escherichia coli 16S RNA or yeast 18S RNA and from Bacillus subtilis tRNA cross-linked to Escherichia coli 16S RNA. The site of cross-linking was unambiguously localized to C1400 in Escherichia coli 16S RNA and to the equivalent position, C1626, in yeast 18S RNA. Direct evidence was also provided for the participation of the wobble base (c)mo5U34 of the tRNA in the cross-link. Furthermore, our results exclude the possibility of minor cross-linking sites at other positions. This new method is reliable, rapid, and easy to handle and should be applicable to any cleavable covalent RNA-RNA duplex. Furthermore, it is sensitive to certain aspects of the steric conformation of such covalent duplexes.

Bacillus subtilis↗
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↗
PubMed1981

The sequence of the ribosomal 16S RNA from Proteus vulgaris. Sequence comparison with E. coli 16S RNA and its use in secondary model building.

The complete nucleotide sequence of the 16S RNA from Proteus vulgaris has been determined. The molecule (1544 nucleotides) shows 93% homology with the sequence of E. coli 16S RNA. Six methylated nucleotides have been localized in positions homologous to those observed in the E. coli RNA molecule. Both E. coli and P. vulgaris 16S RNA chains can be folded up into a common secondary structure scheme. Comparative sequence analysis of the two molecules has provided a valuable contribution to 16S RNA secondary structure model building.

Base Sequence↗
PubMed1981

Structural organization of the 16S ribosomal RNA from E. coli. Topography and secondary structure.

Extensive studies in our laboratory using different ribonucleases resulted in valuable data on the topography of the E.coli 16S ribosomal RNA within the native 30S subunit, within partially unfolded 30S subunits, in the free state, and in association with individual ribosomal proteins. Such studies have precise details on the accessibility of certain residues and delineated highly accessible RNA regions. Furthermore, they provided evidence that the 16S rRNA is organized in its subunit into four distinct domains. A secondary structure model of the E.coli 16S rRNA has been derived from these topographical data. Additional information from comparative sequence analyses of the small ribosomal subunit RNAs from other species sequenced so far has been used.

Computers↗
PubMed1981

A general secondary-structure model for procaryotic and eucaryotic RNAs from the small ribosomal subunits.

A consensus on the folding of the Escherichia coli 16-S ribosomal RNA is emerging and several complete nucleotide sequences of small ribosomal subunit RNAs, covering diverse types of organisms and organelles, are now available. We therefore investigated the extent of both nucleotide sequence and secondary structure conservation that may exist between the E. coli 16-S RNA and other ribosomal RNAs. All the RNA molecules examined could be folded into secondary structure schemes that illustrated remarkable preservation of many structural motifs as well as striking nucleotide sequence conservation compared with the E. coli molecule. This study presents a unitary scheme for the structural organization of the small ribosomal subunit RNAs. The evolutionary constraints on both primary and secondary structures most likely reveal the basic role of some restricted RNA regions in the function of the ribosome.

Animals↗
PubMed1980

[Secondary and topographic structure of ribosomal RNA 16S of Escherichia coli].

We present a model for the secondary structure of 16S ribosomal RNA from E. coli. This model has been deduced by restricting the total number of theoretical base pairings using the following criteria: (1) susceptibility of residues towards enzymatic probes that are specific for either paired or single stranded regions; (2) reactivity of certain residues to chemical modification; (3) evidence for medium and long range interactions; (4) comparative analysis of ribosomal RNA sequences from other organisms.

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↗
PubMed1980

The topography of the 5' end of 16-S RNA in the presence and absence of ribosomal proteins S4 and S20.

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-s RNA in the absence of ribosomal protein. These data yield important information on the topography and organisation of the 5' third of the 16-s RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA.

Base Sequence↗
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↗