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B Ehresmann

Publications and source records attributed to B Ehresmann.

At least 127 records · Page 7Linked to original sources
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

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

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↗
PubMed1989

Crosslinking of transcription factor TFIIIA to ribosomal 5S RNA from X. laevis by trans-diamminedichloroplatinum (II).

Trans-diamminnedichloroplatinum (II) was used to induce reversible crosslinks between 5S rRNA and TFIIIA within the 7S RNP particle from X. laevis immature oocyte. The crosslinked fragments have been unambiguously identified. These fragments exclusively arise from three RNA regions centered around the hinge region at the junction of the three helical domains. Major crosslinking sites are located in region 9-21 (comprising loops A and helix II) and region 54-71 (comprising loop B, helices II and V). A minor site is also found in the 3' part of helix I and helix V (region 100-120). Our results point to the crucial role of the junction region and of the three-dimensional folding of the RNA in the recognition of the 5S rRNA by TFIIIA.

Animals↗
PubMed1989

The solution structure of the Escherichia coli initiator tRNA and its interactions with initiation factor 2 and the ribosomal 30 S subunit.

The conformation of the Escherichia coli initiator tRNA has been investigated using enzymatic and chemical probes. This study was conducted on the naked tRNA and on the tRNA involved in the various steps leading to the formation of the 30 S.IF-2.GTP.fMet-tRNA.AUG complex. A three-dimensional model of the initiator tRNA is presented, which displays several differences with yeast tRNAPhe: (i) the anticodon arm is more rigid; (ii) the presence of an additional nucleotide in the D loop results in specific features in both T and D loops; (iii) C1 and A72 might form a noncanonical base pair. Aminoacylation and formylation induce subtle conformational adjustments near the 3' end, the T arm and the D loop. Initiation factor (IF) 2 interacts with a rather limited portion of the tRNA, covering the T loop and the minor groove of the T stem, and induces an increased flexibility in the anticodon arm. The specific structural features observed in the T loop are probably recognized by IF-2. In the 30 S.IF-2.GTP.fMet-tRNA.AUG complex, additional protections are observed in the acceptor stem and in the anticodon arm, resulting from a strong steric hindrance and from the codon-anticodon interaction within the subunit decoding site.

Base Sequence↗
PubMed1989

Probing the phosphates of the Escherichia coli ribosomal 16S RNA in its naked form, in the 30S subunit, and in the 70S ribosome.

Ethylnitrosourea is an alkylating reagent which preferentially modifies phosphates in nucleic acids. It was used to map phosphates in naked Escherichia coli 16S rRNA engaged in tertiary interactions through hydrogen bonds or ion coordination. Of the phosphates, 7% are found involved in such interactions, and 57% of them are located in loops or interhelical regions, where they are involved in maintaining local intrinsic structures or long-distance tertiary interactions. The other phosphates (43%) are found in helical regions. These phosphates often occur at the proximity of bulged nucleotides or in irregular helices containing noncanonical base pairs (and bulges) and are assumed to bind cations in order to neutralize negative charges and to stabilize unusual phosphate backbone folding. In the 30S subunit, ENU allowed mapping of phosphates in contact with proteins. The RNA is not uniformly engaged in RNA/protein interactions. Regions 1-51, 250-310, 567-612, 650-670, and 1307-1382 are particularly buried whereas the 3'-terminal domain and the 5'-proximal region (nucleotides 53-218) are exposed. The conformation of 16S rRNA is not drastically affected by protein binding, but conformational adjustments are detected in several defined regions. They are found in the 5' domain (region 147-172), in the central domain (region 827-872), in the 3' major domain (nucleotides 955-956, 994, 1054, 1181, 1257, and 1262-1263), and in the 3'-terminal domain (around 1400). The 50S subunit shields clusters of phosphates located at the subunit interface. The most extensive protections are observed in the 3'-terminal domain (1490-1542), in the central region of the molecule (770-930), and in the upper 3' major domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1989

Computer modeling from solution data of spinach chloroplast and of Xenopus laevis somatic and oocyte 5 S rRNAs.

Detailed atomic models of a eubacterial 5 S rRNA (spinach chloroplast 5 S rRNA) and of a eukaryotic 5 S rRNA (somatic and oocyte 5 S rRNA from Xenopus laevis) were built using computer graphic. Both models integrate stereochemical constraints and experimental data on the accessibility of bases and phosphates towards several structure-specific probes. The base sequence was first inserted on to three-dimensional structural fragments picked up in a specially devised databank. The fragments were modified and assembled interactively on an Evans & Sutherland PS330. Modeling was finalized by stereochemical and energy refinement. In spite of some uncertainty in the relative spatial orientation of the substructures, the broad features of the models can be generalized and several conclusions can be reached: (1) both models adopt a distorted Y-shape structure, with helices B and D not far from colinearity; (2) no tertiary interactions exist between loop c and region d or loop e; (3) the internal loops, in particular region d, contain several non-canonical base-pairs of A.A, U.U and A.G types; (4) invariant residues appear to be more important for protein or RNA binding than for maintaining the tertiary structure. The models are corroborated by footprinting experiments with ribosomal proteins and by the analysis of various mutants. Such models help to clarify the structure-function relationship of 5 S rRNA and are useful for designing site-directed mutagenesis experiments.

Animals↗
PubMed1989

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

In a preceding paper [(1987) Nucleic Acids Res. 15, 5787-5801], we have used trans-diamminedichloroplatinum (II) to induce reversible RNA-protein crosslinks within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex and have identified two crosslinking sites on the tRNA. The aim of the present paper is to determine the crosslinking sites on EF-Tu. Two tryptic peptides located in domain I could be identified, a major one (residues 45-74) and a minor one (residues 117-154). The use of Staphylococcus aureus V8 protease led to the isolation of two major peptides (residues 56-68 and 64-68) and one minor peptide (118-124). These results are discussed in the light of the current knowledge of the topography of the EF-Tu/tRNA complex.

Amino Acid Sequence↗
PubMed1989

Use of lead(II) to probe the structure of large RNA's. Conformation of the 3' terminal domain of E. coli 16S rRNA and its involvement in building the tRNA binding sites.

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3' domain of the E. coli 16S rRNA (nucleotides 1295-1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational adjustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337, 1400, 1500 and near the 3' end of the RNA. The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399-1400, 1491-1492 and 1505) protections. A third class of protection (1344-1345, 1393-1395, 1403-1409, 1412-1414, 1504, 1506-1507 and 1517-1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.

Base Sequence↗
PubMed1988

Higher order structure of chloroplastic 5S ribosomal RNA from spinach.

The secondary and tertiary structure of chloroplastic 5S ribosomal RNA from spinach was investigated by the use of several chemical and enzymatic structure probes. The four bases were monitored at one of their Watson-Crick base-pairing positions with dimethyl sulfate [at A(N1) and C(N3)] and with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate [at G(N1) and U(N3)]. Position N7 of purines was probed with diethyl pyrocarbonate (adenines) and with dimethyl sulfate (guanines). Ethylnitrosourea was used to probe phosphate involved in tertiary interaction or in cation coordination. In order to estimate the degree of stability of helices, the various chemical reagents were employed under "native" conditions (300 mM KCl and 20 mM magnesium at 37 degrees C), under "semidenaturing" conditions [1 mM ethylenediaminetetraacetic acid (EDTA) at 37 degrees C], and under denaturing conditions (1 mM EDTA at 90 degrees C). Unstructured regions were also tested with single-strand-specific nucleases T1, U2, and S1 and double-stranded or stacked regions with RNase V1 from cobra Naja naja oxiana venom. The results confirm the existence of the five helices and the two external loops proposed in the consensus model of 5S rRNA. However, the regions depicted as unpaired internal loops appear to be folded into a more complex conformation. A three-dimensional model derived from the present data and graphic modeling for a region encompassing helix IV, helix V, loop D, and loop E (nucleotides 70-110) is proposed. Nucleotides in the so-called loop E (73-79/100-106) display unusual features: Noncanonical base pairs (A-A and A-G) are formed, and three nucleotides (C75, U78, and U105) are bulging out. This region adopts an unwound and extended conformation that can be well suited for tertiary interactions or for protein binding. Several bases and phosphates candidate for the tertiary folding of the RNA were also identified.

Base Composition↗
PubMed1988

The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein.

We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding.

Base Sequence↗
PubMed1988

A comparison of the solution structures and conformational properties of the somatic and oocyte 5S rRNAs of Xenopus laevis.

The secondary and tertiary structures of Xenopus oocyte and somatic 5S rRNAs were investigated using chemical and enzymatic probes. The accessibility of both RNAs towards single-strand specific nucleases (T1, T2, A and S1) and a helix-specific ribonuclease from cobra venom (RNase V1) was determined. The reactivity of nucleobase N7, N3 and N1 positions towards chemical probes was investigated under native (5 mM MgCl2, 100 mM KCl, 20 degrees C) and semi-denaturing (1 mM EDTA, 20 degrees C) conditions. Ethylnitrosourea was used to identify phosphates not reactive towards alkylation under native conditions. The results obtained confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. The chemical reactivity data indicate that loops C and D are involved in a number of tertiary interactions, and loop E folds into an unusual secondary structure. A comparison of the data obtained for the two types of Xenopus 5S rRNA indicates that the conformations of the oocyte and somatic 5S rRNAs are very similar. However, the data obtained with nucleases under native conditions, and chemical probes under semi-denaturing conditions, reveal that helices III and IV in the somatic 5S rRNA are less stable than the same structures in oocyte 5S rRNA. Using chimeric 5S rRNAs, it was possible to demonstrate that the relative resistance of oocyte 5S rRNA to partial denaturation in 4 M urea is conferred by the five oocyte-specific nucleotide substitutions in loop B/helix III. In contrast, the superior stability of oocyte 5S rRNA in the presence of EDTA is related to a single C substitution at position 79.

Animals↗
PubMed1988

Crosslinking of ribosomal protein S18 to 16 S RNA in E.coli ribosomal 30 S subunits by the use of a reversible crosslinking agent: trans-diamminedichloroplatinum(II).

We have previously developed [(1987) Biochemistry 26, 5200-5208] the use of trans-diamminedichloroplatinum(II) to induce reversible RNA-protein crosslinks in the ribosomal 30 S subunit. Protein S18 and, to a lesser extent, proteins S13/S14, S11, S4 and S3 could be crosslinked to the 16 S rRNA. The aim of the present work was to identify the crosslinking sites of protein S18. Three sites could be detected: a major one located in region 825-858, and two others located in regions 434-500 and 233-297. This result is discussed in the light of current knowledge of the topographical localization of S18 in the 30 S subunit and of its relation with function.

Base Sequence↗
PubMed1988

Translational control in E. coli: the case of threonyl-tRNA synthetase.

Genetic studies have shown that expression of the E. coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level. A region called the operator, located 110 nucleotides downstream of the 5' end of the mRNA and between 10 and 50 bp upstream of the translational initiation codon in the thrS gene, is directly involved in that control. The conformation of an in vitro RNA fragment extending over the thrS regulatory region has been investigated with chemical and enzymatic probes. The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA. The conformation of 3 constituent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation. However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region. Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1988

Messenger RNA structure and gene regulation at the translational level in Escherichia coli: the case of threonine:tRNAThr ligase.

Previous work showed that the expression of the Escherichia coli threonine:tRNAThr ligase (EC 6.1.1.3)-encoding gene (thrS) is negatively autoregulated at the translational level and that a region called the operator that is located between 10 and 50 base pairs upstream of the translation initiation codon of the thrS gene is directly involved in that control. The conformation of an in vitro synthesized RNA fragment extending over the thrS regulatory region has been investigated using chemical and enzymatic probes. This study shows that the RNA folds into four well-defined secondary-structure domains, one of them displaying structural similarities to the anticodon arm of tRNAThr. The conformation of three constitutive mutants containing single base changes in the operator region leading to the loss of the regulatory control was also investigated. The replacement of a base in the anticodon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in the regulatory process. However, single mutations in or close to the anticodon-like stem result in a partial or complete reorganization of the structure of the operator region. These rearrangements should affect the binding of the ligase to the operator, leading to loss of the regulatory process.

Amino Acyl-tRNA Synthetases↗