Biomedical subjects

C Ehresmann

Publications and source records attributed to C Ehresmann.

At least 37 records · Page 2Linked to original sources

PubMed2000

Dynamics of the HIV-1 reverse transcription complex during initiation of DNA synthesis.

Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA(3)(Lys) and reverse transcriptase (RT). The vRNA and the primer tRNA(3)(Lys) form several intermolecular interactions in addition to annealing of the primer 3' end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA.tRNA(3)(Lys) complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.

Anticodon↗

PubMed2000

In vivo selection of functional ribosomes with variations in the rRNA-binding site of Escherichia coli ribosomal protein S8: evolutionary implications.

The highly conserved nature of rRNA sequences throughout evolution allows these molecules to be used to build philogenic trees of different species. It is unknown whether the stability of specific interactions and structural features of rRNA reflects an optimal adaptation to a functional task or an evolutionary trap. In the work reported here, we have applied an in vivo selection strategy to demonstrate that unnatural sequences do work as a functional replacement of the highly conserved binding site of ribosomal protein S8. However, growth competition experiments performed between Escherichia coli isolates containing natural and unnatural S8-binding sites showed that the fate of each isolate depended on the growth condition. In exponentially growing cells, one unnatural variant was found to be equivalent to wild type in competition experiments performed in rich media. In culture conditions leading to slow growth, however, cells containing the wild-type sequence were the ultimate winner of the competition, emphasizing that the wild-type sequence is, in fact, the most fit solution for the S8-binding site.

Base Sequence↗

PubMed2000

In vitro evidence for the interaction of tRNA(3)(Lys) with U3 during the first strand transfer of HIV-1 reverse transcription.

Over the course of its evolution, HIV-1 has taken maximum advantage of its tRNA(3)(Lys)primer by utilizing it in several steps of reverse transcription. Here, we have identified a conserved nonanucleotide sequence in the U3 region of HIV-1 RNA that is complementary to the anticodon stem of tRNA(3)(Lys). In order to test its possible role in the first strand transfer reaction, we applied an assay using a donor RNA corresponding to the 5'-part and an acceptor RNA spanning the 3'-part of HIV-1 RNA. In addition, we constructed two acceptor RNAs in which the nonanucleotide sequence complementary to tRNA(3)(Lys)was either substituted (S) or deleted (Delta). We used either natural tRNA(3)(Lys)or an 18 nt DNA as primer and measured the efficiency of (-) strand strong stop DNA transfer in the presence of wild-type, S or Delta acceptor RNA. Mutations in U3 did not decrease the transfer efficiency when reverse transcription was primed with the 18mer DNA. However, they significantly reduced the strand transfer efficiency in the tRNA(3)(Lys)-primed reactions. This reduction was also observed in the presence of nucleocapsid protein. These results suggest that tRNA(3)(Lys)increases (-) strand strong stop transfer by interacting with the U3 region of the genomic RNA. Sequence comparisons suggest that such long range interactions also exist in other lentiviruses.

Base Sequence↗

PubMed2000

Convergence of natural and artificial evolution on an RNA loop-loop interaction: the HIV-1 dimerization initiation site.

Loop-loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop-loop interaction at the dimerization initiation site (DIS), a hairpin located in the 5' noncoding region that contains an autocomplementary sequence in the loop. Only two major DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop-loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization-competent sequences. Surprisingly, increasing interloop complementarity above a threshold of 6 bp did not enhance dimerization, although the combinations of nucleotides forming the theoretically most stable hexanucleotide duplexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs closing the loop and covariations between flanking and central loop nucleotides. Degeneration of the entire loop yielded a complex population of dimerization-competent sequences whose consensus sequence resembles that of wild-type HIV-1. We conclude from these findings that the DIS has evolved to satisfy simultaneous constraints for optimal dimerization affinity and the capacity for homodimerization. Furthermore, the most constrained features of the DIS identified by our experiments could be the basis for the rational design of DIS-targeted antiviral compounds.

Codon, Initiator↗

PubMed2000

The crystal structure of HIV reverse-transcription primer tRNA(Lys,3) shows a canonical anticodon loop.

We have solved to 3.3 A resolution the crystal structure of the HIV reverse-transcription primer tRNA(Lys,3). The overall structure is exactly comparable to the well-known L-shape structure first revealed by yeast tRNA(Phe). In particular, it unambiguously shows a canonical anticodon loop. This contradicts previous results in short RNA fragment studies and leads us to conclude that neither frameshifting specificities of tRNA(Lys) nor tRNA(Lys,3) primer selection by HIV are due to a specific three-dimensional anticodon structure. Comparison of our structure with the results of an NMR study on a hairpin representing a nonmodified anticodon stem-loop makes plausible the conclusion that chemical modifications of the wobble base U34 to 5-methoxycarbonyl-methyl-2-thiouridine and of A37 to 2-methylthio-N-6-threonylcarbamoyl-adenosine would be responsible for a canonical 7-nt anticodon-loop structure, whereas the unmodified form would result in a noncanonical UUU short triloop. The hexagonal crystal packing is remarkable and shows tight dimers of tRNAs forming a right-handed double superhelix. Within the dimers, the tRNAs are associated head-to-tail such that the CCA end of one tRNA interacts with the anticodon of the symmetry-related tRNA. This provides us with a partial view of a codon-anticodon interaction and gives insights into the positioning of residue 37, and of its posttranscriptional modifications, relative to the first base of the codon.

Animals↗

PubMed2000

NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription.

Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer. Some of the modified nucleotides carried by this tRNA must play a key role in the initiation of this process, because unmodified tRNA produced in vitro is only marginally active as primer. To provide a better understanding of the contribution of base modifications in the initiation complex, we have designed a recombinant system that allows tRNA(Lys)3 expression in Escherichia coli. Because of their high level of overexpression, some modifications are incorporated at substoichiometric levels. We have purified the two major recombinant tRNA(Lys)3 subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical techniques. Both species carry psis, Ds, T, t6A, and m7G. Differences are observed at position 34, within the anticodon. One fraction lacks the 5-methylaminomethyl group, whereas the other lacks the 2-thio group. Although the s2U34-containing recombinant tRNA is a less efficient primer, it presents most of the characteristics of the mammalian tRNA. On the other hand, the mnm5U34-containing tRNA has a strongly reduced activity. Our results demonstrate that the modifications that are absent in E. coli (m2G10, psi27, m5C48, m5C49, and m1A58) as well as the mnm5 group at position 34 are dispensable for initiation of reverse transcription. In contrast, the 2-thio group at position 34 seems to play an important part in this process.

Base Sequence↗

PubMed2000

An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment.

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex. We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex. This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.

Bacterial Proteins↗

PubMed2000

Crystal structure of the S15-rRNA complex.

In bacterial ribosomes, the small (30S) ribosomal subunit is composed of 16S rRNA and 21 distinct proteins. Ribosomal protein S15 is of particular interest because it binds primarily to 16S rRNA and is required for assembly of the small subunit and for intersubunit association, thus representing a key element in the assembly of a whole ribosome. Here we report the 2.8 ¿ resolution crystal structure of the highly conserved S15-rRNA complex. Protein S15 interacts in the minor groove with a G-U/G-C motif and a three-way junction. The latter is constrained by a conserved base triple and stacking interactions, and locked into place by magnesium ions and protein side chains, mainly through interactions with the unique three-dimensional geometry of the backbone. The present structure gives insights into the dual role of S15 in ribosome assembly and translational regulation.

Amino Acid Sequence↗

PubMed1999

The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges.

BACKGROUND: An important step in retroviral replication is dimerization of the genomic RNA prior to encapsidation. Dimerization is initiated by the formation of a transient 'kissing-loop complex' that is thought to be subsequently matured into an extended duplex by the nucleocapsid protein (NCp). Although chemical probing and nuclear magnetic resonance spectroscopy have provided insight into the structure of the kissing-loop structure, no structural information concerning the extended-duplex state is available so far. RESULTS: The structure of a minimal HIV-1 RNA dimerization initiation site has been solved at 2.3 A resolution in two different space groups. It reveals a 22 base pair extended duplex with two noncanonical Watson-Crick-like G-A mismatches, each adjacent to a bulged-out adenine. The structure shows significant asymmetry in deep groove width and G-A base-pair conformations. A network of eight magnesium cations was clearly identified, one being unusually chelated by the 3' phosphate of each bulge across an extremely narrowed deep major groove. CONCLUSIONS: These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.

Adenine↗

PubMed1999

Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting.

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.

Amino Acid Sequence↗

PubMed1999

The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site.

E. coli threonyl-tRNA synthetase (ThrRS) is a class II enzyme that represses the translation of its own mRNA. We report the crystal structure at 2.9 A resolution of the complex between tRNA(Thr) and ThrRS, whose structural features reveal novel strategies for providing specificity in tRNA selection. These include an amino-terminal domain containing a novel protein fold that makes minor groove contacts with the tRNA acceptor stem. The enzyme induces a large deformation of the anticodon loop, resulting in an interaction between two adjacent anticodon bases, which accounts for their prominent role in tRNA identity and translational regulation. A zinc ion found in the active site is implicated in amino acid recognition/discrimination.

Amino Acyl-tRNA Synthetases↗

PubMed1999

Structural basis for the specificity of the initiation of HIV-1 reverse transcription.

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition of the viral genome, tRNA3Lys, which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between HIV-1 RNA and tRNA3Lys, but remains poorly understood at the three-dimensional level. We used chemical probing to gain insight into the three-dimensional structure of the viral RNA-tRNA3Lys complex, and enzymatic footprinting to delineate regions interacting with RT. These and previous experimental data were used to derive a three-dimensional model of the initiation complex. The viral RNA and tRNA3Lys form a compact structure in which the two RNAs fold into distinct structural domains. The extended interactions between these molecules are not directly recognized by RT. Rather, they favor RT binding by preventing steric clashes between the nucleic acids and the polymerase and inducing a viral RNA-tRNA3Lys conformation which fits perfectly into the nucleic acid binding cleft of RT. Recognition of the 3' end of tRNA3Lys and of the first template nucleotides by RT is favored by a kink in the template strand promoted by the short junctions present in the previously established secondary structure.

Base Sequence↗

PubMed1999

Crystallization of the dimerization-initiation site of genomic HIV-1 RNA: preliminary crystallographic results.

The genomic RNA of all retroviruses is encapsidated in virions as a dimer of single-stranded chains held together near their 5'-end. For HIV-1, the initial site of dimerization has been shown to be a hairpin with a nine-residue loop containing a self-complementary sequence of six residues. This structure is proposed to promote dimerization by loop-loop interaction and formation of a so-called 'kissing complex'. A 23-nucleotide RNA strand containing the loop enclosed by a seven base-pair stem has been synthesized. This oligomer was crystallized by the vapour-diffusion method at 310 K, pH 6.5, with methyl-pentanediol as the precipitant agent in the presence of MgCl2, KCl and spermine. Quasi-complete diffraction data were obtained at 2.7 A resolution with a conventional X-ray source and at 2.3 A resolution on a synchrotron beamline. The space group is P3121 or its enantiomorph P3221, with cell parameters a = b = 60. 1, c = 65.9 A at ambient temperature, or a = b = 59.0, c = 64.3 A in a nitrogen-gas stream. There are two oligomers per asymmetric unit as determined from absorbance measurements of a dissolved crystal whose volume was carefully determined. In some cases, either perfectly or partially twinned crystals were obtained. Perfect twinning is detected by an apparent hexagonal symmetry and yields unusable crystallographic data, whilst partial twinning yields usable data after adequate processing. Structure solution is under way by searching for heavy-atom derivatives and systematically substituting bromo- or iodo-uridines for uridines.

Base Sequence↗

PubMed1999

Dimerization of HIV-1 genomic RNA of subtypes A and B: RNA loop structure and magnesium binding.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.

Base Sequence↗

PubMed1999

The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site.

The formation of an infectious retrovirus particle requires several RNA-RNA interaction events. In particular, the genomic RNA molecules form a dimeric structure, and a cellular tRNA molecule is annealed to an 18-base complementary region (the primer binding site, or PBS) on the genomic RNA, where it will serve as primer for reverse transcription. tRNAs normally possess a highly stable secondary and tertiary structure; it seems unlikely that annealing of a tRNA molecule to the PBS, which involves unwinding of this structure, could occur efficiently at physiological temperatures without the assistance of a cofactor. Many prior studies have shown that the viral nucleocapsid (NC) protein can act as a nucleic acid chaperone (i.e., facilitate annealing events between nucleic acids), and the assays used to demonstrate this activity include its ability to catalyze dimerization of transcripts representing retroviral genomes and the annealing of tRNA to the PBS in vitro. However, mature NC is not required for these events in vivo, since protease-deficient viral mutants, in which NC is not cleaved from the parental Gag polyprotein, are known to contain dimeric RNAs with tRNA annealed to the PBS. In the present experiments, we have tested recombinant human immunodeficiency virus type 1 Gag polyprotein for nucleic acid chaperone activity. The protein was positive by all of our assays, including the ability to stimulate dimerization and to anneal tRNA to the PBS in vitro. In quantitative experiments, its activity was approximately equivalent on a molar basis to that of NC. Based on these results, we suggest that the Gag polyprotein (presumably by its NC domain) catalyzes the annealing of tRNA to the PBS during (or before) retrovirus assembly in vivo.

Animals↗

PubMed1998

Contacts between reverse transcriptase and the primer strand govern the transition from initiation to elongation of HIV-1 reverse transcription.

HIV-1 reverse transcriptase (RT) utilizes RNA oligomers to prime DNA synthesis. The initiation of reverse transcription requires specific interactions between HIV-1 RNA, primer tRNA3Lys, and RT. We have previously shown that extension of an oligodeoxyribonucleotide, a situation that mimicks elongation, is unspecific and differs from initiation by the polymerization rate and dissociation rate of RT from the primer-template complex. Here, we used replication intermediates to analyze the transition from the initiation to the elongation phases. We found that the 2'-hydroxyl group at the 3' end of tRNA had limited effects on the polymerization and dissociation rate constants. Instead, the polymerization rate increased 3400-fold between addition of the sixth and seventh nucleotide to tRNA3Lys. The same increase in the polymerization rate was observed when an oligoribonucleotide, but not an oligodeoxyribonucleotide, was used as a primer. In parallel, the dissociation rate of RT from the primer-template complex decreased 30-fold between addition of the 17th and 19th nucleotide to tRNA3Lys. The polymerization and dissociation rates are most likely governed by interactions of the primer strand with helix alphaH in the p66 thumb subdomain and the RNase H domain of RT, respectively.

Animals↗

PubMed1998

Solution studies of the dimerization initiation site of HIV-1 genomic RNA.

Dimerization of HIV-1 genomic RNA is an essential step of the viral cycle, initiated at a conserved stem-loop structure which forms a 'kissing complex' involving loop-loop interactions (dimerization initiation site, DIS). A 19mer RNA oligonucleotide (DIS-19) has been synthesized which forms a stable symmetrical dimer in solution at millimolar concentrations. Dimerization does not depend on addition of Mg2+. RNA ligation experiments unambiguously indicate that the formed dimer is a stable kissing complex under the NMR experimental conditions.1H NMR resonance assignments were obtained for DIS-19 at 290 K and pH 6.5. Analysis of the pattern of NOE connectivities reveals that the helix formed by loop-loop base pairing is stacked onto the two terminal stems. Non-canonical base pairs between two essential and conserved adenines are found at the junctions between the two intramolecular and the single intramolecular helices.

Binding Sites↗

PubMed1998

Crystal structure of ribosomal protein S8 from Thermus thermophilus reveals a high degree of structural conservation of a specific RNA binding site.

S8 is one of the core ribosomal proteins. It binds to 16 S RNA with high affinity and independently of other ribosomal proteins. It also acts as a translational repressor in Escherichia coli by binding to its own mRNA. The structure of Thermus thermophilus S8 has been determined by the method of multiple isomorphous replacement at 2.9 A resolution and refined to a crystallographic R-factor of 16.2% (Rfree 27.5%). The two domains of the structure have an alpha/beta fold and are connected by a long protruding loop. The two molecules in the asymmetric unit of the crystal interact through an extensive hydrophobic core and form a tightly associated dimer, while symmetry-related molecules form a joint beta-sheet of mixed type. This type of protein-protein interaction could be realized within the ribosomal assembly. A comparison of the structures of T. thermophilus and Bacillus stearothermophilus S8 shows that the interdomain loop is eight residues longer in the former and reveals high structural conservation of an extensive region, located in the C-terminal domain. From mutational studies this region was proposed earlier to be involved in specific interaction with RNA. On the basis of these data and on the comparison of the two structures of S8, it is proposed that the three-dimensional structure of specific RNA binding sites in ribosomal proteins is highly conserved among different species.

Amino Acid Sequence↗