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Régulateurs transcriptionnels chez les archées hyperthermophiles et leurs virus : analyse moléculaire, fonctionnelle et génétique

Abstract : In Archaea cells all information processes, including transcription, are performed by the Eukarya-like proteins. While the transcriptional machinery of archaea has been well characterized structurally and functionally, very few information concerning the regulation of its activity is available. By working with both cell (crenarchaeota Sulfolobus islandicus) and viral models, we have performed an in-depth study of three transcriptional regulators: two viral regulators, SvtR and AFV1p06, and a cell regulator Sta1. The obtained results allow to better understand the mechanisms of transcriptional regulation in archaea. Concerning the protein SvtR encoded by the virus SIRV1 that infects S. islandicus, we continued the research project that had identified its structure and function. We were focused on identification and characterization of all of SvtR targets in the viral genome and on the study of the mechanisms of regulation. For this purpose, we established the sequence of consensus site recognized by SvtR using systematic mutagenesis of one of its previously characterized binding sites. This site is present in the promoters of 10 genes meaning that SvtR may regulate the activity of more than 20% of SIRV1 genes. Its targets include all known genes encoding proteins of the viral capsid. Functional analysis of SvtR has demonstrated that, according to the target, this protein is a versatile regulator acting as transcriptional activator or repressor. Taking as a model the gp30 gene promoter, we demonstrated by several approaches that regulation of this promoter includes the polymerization of the protein from its primary binding site towards the TATA-box. Such a mechanism of transcriptional regulation is new in archaea. Second, we performed a structural analysis of the protein AFV1p06 encoded by the virus AFV1 which infects Acidianus hospitalis. The structural analysis of AFV1p06 revealed the presence of a C2H2 zinc finger domain regarded hitherto as specific to eukaryotes. We demonstrated that AFV1p06 has ability to bind specifically to DNA sequences rich in GC. AFV1p06 is the first archaeal DNA binding protein with zinc finger domain characterized in vitro. The third transcriptional regulator, Sta1 is encoded by the genome of Sulfolobales. The protein RadA is able to activate the transcription of viral as well as chromosomal genes in response to DNA damage. To understand its role in the cell, we attempted, without success, to knockout the sta1 gene in S. islandicus RYE15A. This result indicates that the sta1 gene is probably essential. The strain S. islandicus LAL14 /1 is a model strain to study host-virus interaction in archaea. The sequencing of the genome of this strain opened the way to establish a genetic system for this model and allowed us to construct knockout mutants for several LAL14/1 genes (pyrEF-; ΔCRISPR1). Our unsuccessful attempts to inactivate topR2, another candidate gene encoding reverse gyrase indicate that topR2 function could be essential. The construction of the ΔCRISPR1 mutant opens the way to obtain a derivative of LAL14/1 entirely lacking the CRISPR system. Such a mutant will be very useful for the future studies of function and role of CRISPRs in archaea in general but also will allow to verify the hypothesis of involvement of CRISPRs in the phenotype of resistance of LA14/1 to SIRV1. All the results of this thesis contribute to an improved understanding of molecular mechanisms in archaeal cells and their viruses.
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Submitted on : Wednesday, March 26, 2014 - 5:27:46 PM
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Chloe Danioux. Régulateurs transcriptionnels chez les archées hyperthermophiles et leurs virus : analyse moléculaire, fonctionnelle et génétique. Biologie moléculaire. Université Pierre et Marie Curie - Paris VI, 2014. Français. ⟨NNT : 2014PA066010⟩. ⟨tel-00966549⟩

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