DISSECTION DU MÉCANISME DE TERMINAISON/ANTITERMINAISON AU NIVEAU DU TERMINATEUR TRI DU PHAGE LAMBDA : APPLICATION A L'ÉTUDE DES COMPLEXES ARN-PROTÉINE IN VIVO

Abstract : In Escherichia coli transcription termination can occur by two distinct mechanisms: intrinsic termination corresponds to a signal on the template DNA, coding for a GC rich stem-loop structure followed by a strech of U residues on the RNA, that promotes the releasing of the RNA polymerase from the template DNA. The second mechanism is governed by a termination factor called Rho. Rho is responsible for approximately half of the termination processes in E. coli. During my PhD thesis I have focused on this second mechanism, and specially its regulation in vivo (antitermination). Rho acts as a ringshaped hexamer, it binds to the nascent RNA at a Rho loading site (also called RUT), then translocates along the RNA, using its ATP hydrolysis activity and dissiociate the transcription elongation complex stalled at a pause site. Rho dependent terminators have low defined sequences and very few have been analysed in details. The lambda (λ) tR1 is the classical paradigm for Rho-dependant termination that has been well studied both in vitro and in vivo. Rho-dependant transcription termination at he phage λtR1 terminator is governed primarily by the upstream rut element that encode two RNA regions RUTA and RUTB. The two regions are separated by BOXB RNA motif which is believed to be dispensable for Rho activity but serves as a binding site for λ N protein in the antitermination process. By using a minimal in vivo termination system we have shown that the intervening BOXB RNA motif has a double function in the mechamism of termination antitermination at λtR1 that regulate the temporal expression of the phage λ genome. As a folded stem-loop structure, BOXB acts as a hinge that brings RUTA and RUTB side by side for optimal interaction with Rho leading to efficient termination. Conversely, the binding of N protein to BOXB induces antitermination at λtR1 by preventing access of Rho to the rut sequence. This dual role was clearly demonstrated in vivo by substituting the N/BOXB couple with the unrelated coat protein of phage MS2 and its stem-loop RNA target. In addition to this work, I have used this property of a nucleo-protein complex to block transcription termination by Rho, to develop a new and versatile approach for studying, in vivo, RNA-protein complexes.
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https://tel.archives-ouvertes.fr/tel-00009769
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Submitted on : Friday, August 5, 2005 - 4:45:29 PM
Last modification on : Wednesday, June 27, 2018 - 8:24:02 AM
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  • HAL Id : tel-00009769, version 2

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Erwann Vieu. DISSECTION DU MÉCANISME DE TERMINAISON/ANTITERMINAISON AU NIVEAU DU TERMINATEUR TRI DU PHAGE LAMBDA : APPLICATION A L'ÉTUDE DES COMPLEXES ARN-PROTÉINE IN VIVO. domain_other. Université d'Orléans, 2004. Français. ⟨tel-00009769v2⟩

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