Biomedical subjects

Chantal Ehresmann

Publications and source records attributed to Chantal Ehresmann.

15 recordsLinked to original sources

PubMed2003

Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA.

The 16S rRNA-binding ribosomal protein S15 is a key component in the assembly of the small ribosomal subunit in bacteria. We have shown that S15 from the extreme thermophile Thermus thermophilus represses the translation of its own mRNA in vitro, by interacting with the leader segment of its mRNA. The S15 mRNA-binding site was characterized by footprinting experiments, deletion analysis and site-directed mutagenesis. S15 binding triggers a conformational rearrangement of its mRNA into a fold that mimics the conserved three-way junction of the S15 rRNA-binding site. This conformational change masks the ribosome entry site, as demonstrated by direct competition between the ribosomal subunit and S15 for mRNA binding. A comparison of the T.thermophilus and Escherichia coli regulation systems reveals that the two regulatory mRNA targets do not share any similarity and that the mechanisms of translational inhibition are different. Our results highlight an astonishing plasticity of mRNA in its ability to adapt to evolutionary constraints, that contrasts with the extreme conservation of the rRNA-binding site.

Bacterial Proteins↗

PubMed2003

Does the HIV-1 primer activation signal interact with tRNA3(Lys) during the initiation of reverse transcription?

Reverse transcription of HIV-1 RNA is primed by a tRNA3(Lys) molecule bound at the primer binding site (PBS). Complex intermolecular interactions were proposed between tRNA3(Lys) and the RNA of the HIV-1 Mal isolate. Recently, an alternative interaction was proposed between the TPsiC stem of tRNA3(Lys) and a primer activation signal (PAS) of the Lai and Hxb2 RNAs, suggesting major structural variations in the reverse transcription complex of different HIV-1 strains. Here, we analyzed mutants of the Hxb2 RNA that prevent the interaction between the PAS and tRNA3(Lys) or/and a complementary sequence in the viral RNA. We compared the kinetics of reverse transcription of the wild type and mutant Hxb2 RNAs, using either tRNA3(Lys) or an 18mer oligoribonucleotide complementary to the PBS, which cannot interact with the PAS, as primers. We also used chemical probing to test the structure of the mutant and wild type RNAs, as well as the complex formed between the later RNA and tRNA3(Lys). These experiments, together with the analysis of long term replication data of mutant viruses obtained by C. Morrow and coworkers (Birmingham, USA) that use alternate tRNAs as primers, strongly suggest that the interaction between the Hxb2 PAS and tRNA3(Lys) does not exist. Instead, the effects of the vRNA mutations on reverse transcription seem to be linked to incorrect folding of the mutant RNAs.

Base Sequence↗

PubMed2003

Effects of tRNA 3 Lys aminoacylation on the initiation of HIV-1 reverse transcription.

HIV-1 utilizes cellular tRNA(3)(Lys) to prime the initiation of reverse transcription. The selective incorporation of cytoplasmic tRNA(3)(Lys) into HIV-1 particles was recently shown to involve the lysyl-tRNA synthetase, and hence, the encapsidated tRNA(3)(Lys) is likely to be aminoacylated. Here, we tested the effect of aminoacylation on the initiation of reverse transcription. We show that HIV-1 reverse transcriptase is unable to extend lysyl-tRNA(3)(Lys). In addition, the viral polymerase does not significantly enhance the rate of tRNA deacylation, in contrast with previous studies on avian retroviruses. Thus, aminoacylation of the primer tRNA might prevent the initiation of HIV-1 reverse transcription from taking place before viral budding and maturation.

Acetyltransferases↗

PubMed2003

The modular structure of Escherichia coli threonyl-tRNA synthetase as both an enzyme and a regulator of gene expression.

In addition to its role in tRNA aminoacylation, Escherichia coli threonyl-tRNA synthetase is a regulatory protein which binds a site, called the operator, located in the leader of its own mRNA and inhibits translational initiation by competing with ribosome binding. This work shows that the two essential steps of regulation, operator recognition and inhibition of ribosome binding, are performed by different domains of the protein. The catalytic and the C-terminal domain of the protein are involved in binding the two anticodon arm-like structures in the operator whereas the N-terminal domain of the enzyme is responsible for the competition with the ribosome. This is the first demonstration of a modular structure for a translational repressor and is reminiscent of that of transcriptional regulators. The mimicry between the operator and tRNA, suspected on the basis of previous experiments, is further supported by the fact that identical regions of the synthetase recognize both the operator and the tRNA anticodon arm. Based on these results, and recent structural data, we have constructed a computer-derived molecular model for the operator-threonyl-tRNA synthetase complex, which sheds light on several essential aspects of the regulatory mechanism.

Binding Sites↗

PubMed2002

HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics.

Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.

Anti-Bacterial Agents↗

PubMed2002

Direct and indirect contributions of RNA secondary structure elements to the initiation of HIV-1 reverse transcription.

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition between the viral RNA (vRNA), tRNA(3)(Lys), which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between the HIV-1 RNA and tRNA(3)(Lys). Here, we compared the relative importance of the secondary structure elements of this complex in the initiation process. To this aim, we used the previously published three-dimensional model of the initiation complex to rationally introduce a series of deletions and substitutions in the vRNA. When necessary, we used chemical probing to check the structure of the tRNA(3)(Lys)-mutant vRNA complexes. For each of them, we measured the binding affinity of RT and the kinetics of initial extension of tRNA(3)(Lys) and of synthesis of the (-) strand strong stop DNA. Our results were overall in keeping with the three-dimensional model of the initiation complex. Surprisingly, we found that disruption of the intermolecular template-primer interactions, which are not directly recognized by RT, more severely affected reverse transcription than deletions or disruption of one of the intramolecular helices to which RT directly binds. Perturbations of the highly constrained junction between the intermolecular helix formed by the primer binding site and the 3' end of tRNA(3)(Lys) and the helix immediately upstream also had dramatic effects on the initiation of reverse transcription. Taken together, our results demonstrate the overwhelming importance of the overall three-dimensional structure of the initiation complex and identify structural elements that constitute promising targets for anti-initiation-specific drugs.

Base Sequence↗

PubMed2002

Do mRNA and rRNA binding sites of E.coli ribosomal protein S15 share common structural determinants?

Escherichia coli ribosomal protein S15 recognizes two RNA targets: a three-way junction in 16S rRNA and a pseudoknot structure on its own mRNA. Binding to mRNA occurs when S15 is expressed in excess over its rRNA target, resulting in an inhibition of translation start. The sole apparent similarity between the rRNA and mRNA targets is the presence of a G-U/G-C motif that contributes only modestly to rRNA binding but is essential for mRNA. To get more information on the structural determinants used by S15 to bind its mRNA target as compared to its rRNA site, we used site-directed mutagenesis, substitution by nucleotide analogs, footprinting experiments on both RNA and protein, and graphic modeling. The size of the mRNA-binding site could be reduced to 45 nucleotides, without loss of affinity. This short RNA preferentially folds into a pseudoknot, the formation of which depends on magnesium concentration and temperature. The size of the loop L2 that bridges the two stems of the pseudoknot through the minor groove could not be reduced below nine nucleotides. Then we showed that the pseudoknot recognizes the same side of S15 as 16S rRNA, although shielding a smaller surface area. It turned out that the G-U/G-C motif is recognized from the minor groove in both cases, and that the G-C pair is recognized in a very similar manner. However, the wobble G-U pair of the mRNA is not directly contacted by S15, as in rRNA, but is most likely involved in building a precise conformation of the RNA, essential for binding. Otherwise, unique specific features are utilized, such as the three-way junction in the case of 16S rRNA and the looped out A(-46) for the mRNA pseudoknot.

Binding Sites↗

PubMed2002

Primer unblocking and rescue of DNA synthesis by azidothymidine (AZT)-resistant HIV-1 reverse transcriptase: comparison between initiation and elongation of reverse transcription and between (-) and (+) strand DNA synthesis.

Azidothymidine (AZT) is a widely used inhibitor of type 1 human immunodeficiency virus reverse transcriptase (RT) that acts as chain terminator. Upon treatment, mutations conferring AZT resistance to RT are gradually selected. It has been shown that resistant RT is able to unblock the AZT-terminated primer by an ATP-dependent mechanism. However, this resistance mechanism has only been demonstrated for DNA-dependent DNA elongation. Here, we compared the AZT resistance of mutant RT during DNA elongation on DNA and RNA templates. We showed that, during DNA elongation, primer unblocking and rescue of DNA synthesis take place with similar rate constants on DNA and RNA templates. However, the fraction of a primer eventually repaired during RNA-dependent DNA synthesis is 2x lower compared with that of DNA-dependent synthesis, leading to reduced resistance. We also compared the initiation of reverse transcription, which uses tRNA(3)(Lys) as a primer and displays characteristic kinetic features, and the subsequent RNA-dependent elongation. Unlike during elongation, resistant RT was unable to unblock the AZT-terminated primer during initiation of (-) DNA strand synthesis. Our results demonstrate that the efficiency of primer unblocking conferred by the AZT resistance mutations greatly vary during the different steps of the provirus synthesis. These results also suggest that inhibitors specifically targeting the initiation of reverse transcription might prove to be advantageous, as compared with elongation inhibitors.

Base Sequence↗

PubMed2002

RNA loop-loop interactions as dynamic functional motifs.

RNA loop-loop interactions are frequently used to trigger initial recognition between two RNA molecules. In this review, we present selected well-documented cases that illustrate the diversity of biological processes using RNA loop-loop recognition properties. The first one is related to natural antisense RNAs that play a variety of regulatory functions in bacteria and their extra-chromosomal elements. The second one concerns the dimerization of HIV-1 genomic RNA, which is responsible for the encapsidation of a diploid RNA genome. The third one concerns RNA interactions involving double-loop interactions. These are used by the bicoid mRNA to form dimers, a property that appears to be important for mRNA localization in drosophila embryo, and by bacteriophage phi29 pRNA which forms hexamers that participate in the translocation of the DNA genome through the portal vertex of the capsid. Despite the high diversity of systems and mechanisms, some common features can be highlighted. (1) Efficient recognition requires rapid bi-molecular binding rates, regardless of the RNA pairing scheme. (2) The initial recognition is favored by particular conformations of the loops enabling a proper presentation of nucleotides (generally a restricted number) that initiate the recognition process. (3) The fate of the initial reversible loop-loop complex is dictated by both functional and structural constraints. RNA structures have evolved either to "freeze" the initial complex, or to convert it into a more stable one, which involves propagation of intermolecular interactions along topologically feasible pathways. Stabilization of the initial complex may also be assisted by proteins and/or formation of additional contacts.

Animals↗

PubMed2002

Structural and functional properties of the HIV-1 RNA-tRNA(Lys)3 primer complex annealed by the nucleocapsid protein: comparison with the heat-annealed complex.

The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo.

Base Pairing↗

PubMed2002

The HIV-1 Nef protein enhances the affinity of reverse transcriptase for RNA in vitro.

Several viral proteins, including nucleocapsid protein, integrase, Vif, Tat, and Nef have been proposed to act as cofactors of HIV-1 reverse transcription. Using two viral RNA probes, one overlapping the primer-binding site (PBS) and the other representing the ribosomal frameshifting signal (FS) of HIV-1 RNA, we found that recombinant full-length Nef protein (NefLAI) increased the affinity of reverse transcriptase (RT) for RNA in vitro, and interacted directly with RT in protein co-precipitation assays. The effect on RT-RNA binding and the capacity of Nef to interact with RT was also observed with N-terminal deletion mutant NefDelta57 and NefSF2, although to a lesser level. NefDelta57 corresponded to the processed Nef protein present in the internal core of mature virions, and lacked the N-myristoylated N-terminus and N-terminal region implicated in virus infectivity and pathogenicity in vivo. NefSF2, a Nef allele from a highly pathogenic strain of HIV-1, differed from NefLAI by the amino acid sequence and immunoreactivity of its N-terminal domain. The effect observed with NefSF2 and NefDelta57, and data from phage biopanning experiments suggested that the RT-binding region in Nef involved the C-terminal flexible loop of its C-terminal domain, but the function in RT-RNA binding was also influenced by its N-terminal domain.

Amino Acid Sequence↗

PubMed2002

Structural basis of translational control by Escherichia coli threonyl tRNA synthetase.

Escherichia coli threonyl-tRNA synthetase (ThrRS) represses the translation of its own messenger RNA by binding to an operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the mRNA uses the recognition mode of the tRNA anticodon loop to initiate binding. The final positioning of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface. The finding of other thrS operators that have this conserved motif leads to a generalization of this regulatory mechanism to a subset of Gram-negative bacteria.

Anticodon↗

PubMed2002

Secondary structure of the 3' terminus of hepatitis C virus minus-strand RNA.

The 3'-terminal ends of both the positive and negative strands of the hepatitis C virus (HCV) RNA, the latter being the replicative intermediate, are most likely the initiation sites for replication by the viral RNA-dependent RNA polymerase, NS5B. The structural features of the very conserved 3' plus [(+)] strand untranslated region [3' (+) UTR] are well established (K. J. Blight and C. M. Rice, J. Virol. 71:7345-7352, 1997). However, little information is available concerning the 3' end of the minus [(-)] strand RNA. In the present work, we used chemical and enzymatic probing to investigate the conformation of that region, which is complementary to the 5' (+) UTR and the first 74 nucleotides of the HCV polyprotein coding sequence. By combining our experimental data with computer predictions, we have derived a secondary-structure model of this region. In our model, the last 220 nucleotides, where initiation of the (+) strand RNA synthesis presumably takes place, fold into five stable stem-loops, forming domain I. Domain I is linked to an overall less stable structure, named domain II, containing the sequences complementary to the pseudoknot of the internal ribosomal entry site in the 5' (+) UTR. Our results show that, even though the (-) strand 3'-terminal region has the antisense sequence of the 5' (+) UTR, it does not fold into its mirror image. Interestingly, comparison of the replication initiation sites on both strands reveals common structural features that may play key functions in the replication process.

Base Sequence↗

PubMed2001

In vitro evidence for a long range pseudoknot in the 5'-untranslated and matrix coding regions of HIV-1 genomic RNA.

The 5'-untranslated leader region of human immunodeficiency virus type 1 (HIV-1) RNA contains multiple signals that control distinct steps of the viral replication cycle such as transcription, reverse transcription, genomic RNA dimerization, splicing, and packaging. It is likely that fine tuned coordinated regulation of these functions is achieved through specific RNA-protein and RNA-RNA interactions. In a search for cis-acting elements important for the tertiary structure of the 5'-untranslated region of HIV-1 genomic RNA, we identified, by ladder selection experiments, a short stretch of nucleotides directly downstream of the poly(A) signal that interacts with a nucleotide sequence located in the matrix region. Confirmation of the sequence of the interacting sites was obtained by partial or complete inhibition of this interaction by antisense oligonucleotides and by nucleotide substitutions. In the wild type RNA, this long range interaction was intramolecular, since no intermolecular RNA association was detected by gel electrophoresis with an RNA mutated in the dimerization initiation site and containing both sequences involved in the tertiary interaction. Moreover, the functional importance of this interaction is supported by its conservation in all HIV-1 isolates as well as in HIV-2 and simian immunodeficiency virus. Our results raise the possibility that this long range RNA-RNA interaction might be involved in the full-length genomic RNA selection during packaging, repression of the 5' polyadenylation signal, and/or splicing regulation.

5' Untranslated Regions↗

PubMedDate not supplied

The RNA binding protein FMRP: new connections and missing links.

The loss of the fragile X mental retardation protein (FMRP) is responsible for the most common cause of inherited mental retardation called the fragile X syndrome. FMRP is suspected to participate in the synaptic plasticity of neurons by acting on posttranscriptional control of gene expression. FMRP is an RNA binding protein that associates with mRNAs together with other proteins to form large ribonucleoprotein complexes. These complexes are proposed to participate in the transport, localization and translation of target mRNAs. Progress has been made recently in the identification of the mRNAs and the proteins present in these complexes and a possible connection with the micro-RNA dependent regulatory pathway has been established.

Female↗