Indiana University-Purdue University Indianapolis (IUPUI) / In an effort to advance the methodology in analyzing RNAPII protein-protein interaction network and to determine the role of the CTD in controlling RNAPII transcription, we devised a method to specifically isolate the CTD-associated and CTD-less RNAPII to identify the proteins that interact with both the CTD and the globular core of RNAPII using novel purification scheme coupled to quantitative proteomics.

Ce travail de thèse porte sur l’étude des ARN polymérases ARN dépendant impliquées dans le RNA silencing chez Arabidopsis thaliana. Durant ma thèse, la recherche d'interacteurs des RDR, parmi des protéines impliquées dans le RNA silencing, a permis la détection d'interaction entre RDR6 et SDE3, RDR6 et SGS3, mais aussi entre SDE3 et SGS3 en Co-IP et BiFC. Une co-localisation de ces protéines a été observée lorsqu'elles sont produites transitoirement dans des cellules épidermales de N. benthamiana.Un crible d’une banque d’ADNc d’A. thaliana par double hybride de levure, a permis d’isoler des interacteurs potentiels de RDR6. Deux interacteurs potentiels, AtUAP56-1 et U2B’’, sont impliqués dans l’épissage des précurseurs des ARNm. Un effet sur le RNA silencing dans des mutants de l’épissage en 3’ des ARNm était connu et nous avons confirmé l’interaction entre RDR6 et AtUAP56-1 par BiFC. L’étude de lignées mutantes pour AtUAP56-1 a donc été initiée.Une étude biochimique de RDR6 et de RDR2 a été réalisée. Des formes recombinantes de RDR2 et RDR6 ont été produites de façon transitoire dans des feuilles de N. benthamiana, et une étude comparative de RDR2 et RDR6 a été réalisée. Les deux RDR sont actives sur des matrices ARN et ADN, et montrent in vitro une activité amorce-indépendante. De plus, nous avons détecté pour la première fois une activité amorce-dépendante de RDR6 et RDR2. Ces résultats apportent de nouvelles données biochimiques qui sont en accord avec les études menées in vivo et enrichissent les modèles actuels du RNA silencing. / The aim of this work was to study RNA-dependent RNA polymerases involved in RNA silencing in Arabidopsis thaliana. During my thesis, the search for RDR interactors among proteins involved in RNA silencing allowed the detection of interactions between RDR6 and SDE3, RDR6 and SGS3, and also between SDE3 and SGS3 using Co-IP and BiFC. In addition, the co-localisation of these proteins was observed when produced transiently in epidermal cells of N. Benthamiana.A screen of an A. thaliana cDNA library by yeast two hybrid allowed us to identify some putative new RDR6 interactors. Two putative RDR6 interactors, AtUAP56 and U2B’’, are known to be involved in pre-miRNA splicing. Furthermore, a link between pre-mRNA 3’ splicing and RNA silencing was previously reported. We also confirmed the interaction between AtUAP56-1 and RDR6 by BiFC. An investigation of A. thaliana of AtUAP56-1 mutants has been initiated.Recombinant RDRs were produced transiently in N. Benthamiana, and a biochemical comparative study of RDR2 and RDR6 performed. We found that RDR2, like RDR6, has a de novo polymerase activity on DNA and RNA templates, and for both RDRs we also showed, for the first time, a primer-dependant synthesis of dsRNA from RNA template. These findings provide important new insights into our understanding of the molecular mechanisms of RNA silencing amplification in Arabidopsis.

A bactéria Escherichia coli é encontrada em diversos habitats e deve estar preparada para sobreviver e crescer em condições desfavoráveis. A adaptação da bactéria a diferentes condições obriga-a a controlar a expressão de genes de forma eficiente. Uma das formas primárias de controle de expressão gênica é a competição entre os diversos fatores sigma pela ligação ao cerne da RNA polimerase. <font face=\"Symbol\">d70 é o fator sigma mais abundante e participa da transcrição da maioria dos genes de E. coli, enquanto que <font face=\"Symbol\">dS é o segundo em importância e reconhece promotores de genes relacionados à resposta geral ao estresse. O gene rpoS, que codifica para <font face=\"Symbol\">dS é altamente polimórfico, e adquiri mutações frequentemente. A mutação pontual C<font face=\"Symbol\">&#174;T na posição 97 da ORF de rpoS, resulta em um códon de parada TAG (âmbar). Um Shine-Dalgarno alternativo e um códon de início de tradução na posição 157 dá início a uma proteína RpoS truncada, que é parcialmente funcional. Uma das situações de estresse na qual <font face=\"Symbol\">dS é ativado corresponde à privação de fosfato inorgânico. Porém, na limitação deste nutriente ocorre também a ativação do regulon PHO, cujos genes são predominantemente transcritos por <font face=\"Symbol\">d70. Neste trabalho, o efeito da versão truncada de RpoS sobre a expressão de genes dependentes de <font face=\"Symbol\">d70 (lacZ, phoA e pstS) e de genes dependentes de <font face=\"Symbol\">dS (osmY e proU) foi testado. Foram também realizados ensaios de estresse oxidativo, osmótico e pelo frio. O perfil de atividade parcial descrito para RpoSam pôde ser observado em alguns casos, porém em outros o comportamento deste alelo se assemelhou ao do mutante rpoS nulo. Paralelamente, foi testada também uma cepa de E. coli que carrega a mutação âmbar em rpoS, mas esta é suprimida, resultando na expressão de uma proteína RpoS normal. A proteína RpoS truncada não pôde ser visualizada em immunoblots, provavelmente porque esta é traduzida de forma pouco eficiente a partir do Shine-Dalgarno alternativo. Com o objetivo de incrementar a detecção de RpoS em ensaios de imuno-detecção, foi inserida por recombinação alélica uma etiqueta SPA altamente imunogênica na porção C-terminal da proteína. / Escherichia coli can be found in many different habitats and has to be prepared to survive and grow under unfavorable conditions. Bacteria adaptation to different growth conditions requires an efficient control of gene expression. One of the primary forms of gene expression control is the competition between different sigma factors for the binding to the core RNA polymerase. <font face=\"Symbol\">d70 is the most abundant sigma factor and participates in the transcription of most E. coli genes. <font face=\"Symbol\">dS is the second one in importance and recognizes promoters of genes related to the general stress response. The rpoS gene, which encodes <font face=\"Symbol\">dS, is highly polymorphic and acquires mutations very often. The transition C<font face=\"Symbol\">&#174;T at position 97 in the rpoS ORF results in a stop codon TAG (amber). Due to the presence of an alternative Shine-Dalgarno and a translation initiation codon at position 157 a truncated RpoS protein that is partially functional is translated. One of the stress situations that <font face=\"Symbol\">dS is activated is the starvation for inorganic phosphate. Phosphate limitation also triggers the activation of the PHO regulon, whose genes are predominantly transcribed by <font face=\"Symbol\">d70. In the present study, the effect of the truncated version of RpoS on the expression of <font face=\"Symbol\">d70 dependent genes (lacZ, phoA e pstS) and <font face=\"Symbol\">dS dependent genes (osmY e proU) was tested. Bacteria were also assayed for sensitivity to oxidative, osmotic and cold stress. The profile of partial activity described for the truncated RpoS could be observed in some cases, while in others the behavior of this allele resembled the rpoS null mutant. In parallel, an E. coli strain which suppresses the amber mutation in RpoS, resulting in the expression of a normal protein was also tested. A band correponding to the truncated RpoS could not be detected in immunoblots probably due to inefficient translation from the alternative Shine-Dalgarno. To improve the detection of RpoS, a highly immunogenic SPA tag was inserted in the C-terminal region of the protein.

A bactéria Escherichia coli é encontrada em diversos habitats e deve estar preparada para sobreviver e crescer em condições desfavoráveis. A adaptação da bactéria a diferentes condições obriga-a a controlar a expressão de genes de forma eficiente. Uma das formas primárias de controle de expressão gênica é a competição entre os diversos fatores sigma pela ligação ao cerne da RNA polimerase. <font face=\"Symbol\">d70 é o fator sigma mais abundante e participa da transcrição da maioria dos genes de E. coli, enquanto que <font face=\"Symbol\">dS é o segundo em importância e reconhece promotores de genes relacionados à resposta geral ao estresse. O gene rpoS, que codifica para <font face=\"Symbol\">dS é altamente polimórfico, e adquiri mutações frequentemente. A mutação pontual C<font face=\"Symbol\">&#174;T na posição 97 da ORF de rpoS, resulta em um códon de parada TAG (âmbar). Um Shine-Dalgarno alternativo e um códon de início de tradução na posição 157 dá início a uma proteína RpoS truncada, que é parcialmente funcional. Uma das situações de estresse na qual <font face=\"Symbol\">dS é ativado corresponde à privação de fosfato inorgânico. Porém, na limitação deste nutriente ocorre também a ativação do regulon PHO, cujos genes são predominantemente transcritos por <font face=\"Symbol\">d70. Neste trabalho, o efeito da versão truncada de RpoS sobre a expressão de genes dependentes de <font face=\"Symbol\">d70 (lacZ, phoA e pstS) e de genes dependentes de <font face=\"Symbol\">dS (osmY e proU) foi testado. Foram também realizados ensaios de estresse oxidativo, osmótico e pelo frio. O perfil de atividade parcial descrito para RpoSam pôde ser observado em alguns casos, porém em outros o comportamento deste alelo se assemelhou ao do mutante rpoS nulo. Paralelamente, foi testada também uma cepa de E. coli que carrega a mutação âmbar em rpoS, mas esta é suprimida, resultando na expressão de uma proteína RpoS normal. A proteína RpoS truncada não pôde ser visualizada em immunoblots, provavelmente porque esta é traduzida de forma pouco eficiente a partir do Shine-Dalgarno alternativo. Com o objetivo de incrementar a detecção de RpoS em ensaios de imuno-detecção, foi inserida por recombinação alélica uma etiqueta SPA altamente imunogênica na porção C-terminal da proteína. / Escherichia coli can be found in many different habitats and has to be prepared to survive and grow under unfavorable conditions. Bacteria adaptation to different growth conditions requires an efficient control of gene expression. One of the primary forms of gene expression control is the competition between different sigma factors for the binding to the core RNA polymerase. <font face=\"Symbol\">d70 is the most abundant sigma factor and participates in the transcription of most E. coli genes. <font face=\"Symbol\">dS is the second one in importance and recognizes promoters of genes related to the general stress response. The rpoS gene, which encodes <font face=\"Symbol\">dS, is highly polymorphic and acquires mutations very often. The transition C<font face=\"Symbol\">&#174;T at position 97 in the rpoS ORF results in a stop codon TAG (amber). Due to the presence of an alternative Shine-Dalgarno and a translation initiation codon at position 157 a truncated RpoS protein that is partially functional is translated. One of the stress situations that <font face=\"Symbol\">dS is activated is the starvation for inorganic phosphate. Phosphate limitation also triggers the activation of the PHO regulon, whose genes are predominantly transcribed by <font face=\"Symbol\">d70. In the present study, the effect of the truncated version of RpoS on the expression of <font face=\"Symbol\">d70 dependent genes (lacZ, phoA e pstS) and <font face=\"Symbol\">dS dependent genes (osmY e proU) was tested. Bacteria were also assayed for sensitivity to oxidative, osmotic and cold stress. The profile of partial activity described for the truncated RpoS could be observed in some cases, while in others the behavior of this allele resembled the rpoS null mutant. In parallel, an E. coli strain which suppresses the amber mutation in RpoS, resulting in the expression of a normal protein was also tested. A band correponding to the truncated RpoS could not be detected in immunoblots probably due to inefficient translation from the alternative Shine-Dalgarno. To improve the detection of RpoS, a highly immunogenic SPA tag was inserted in the C-terminal region of the protein.

This dissertation is divided into two main sections describing major portions of my Ph.D. research: (1) development of two enzymatic assays for identifying inhibitors of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and the associated proofreading exonuclease complexes, two key enzymatic activities of SARS-CoV-2, the virus responsible for the COVID-19 pandemic and (2) the design and implementation of four novel single-color fluorescent DNA sequencing by synthesis (SBS) methods, including the synthesis of many of the key nucleotide analogues required for these studies. In response to the COVID-19 pandemic, the first part of my research is focused on the discovery of potential therapeutics for combating coronavirus infections. Chapter 1 describes the identification of several polymerase and exonuclease inhibitors for SARS-CoV-2 using novel mass spectrometry-based molecular assays. SARS-CoV-2 has an exonuclease complex, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RdRp and the exonuclease could overcome this deficiency. Chapter 1 reports the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of identified exonuclease inhibitors, RNAs terminated with the active forms of the prodrugs like Sofosbuvir, Remdesivir and Favipiravir were largely protected from excision by the exonuclease, while in the absence of exonuclease inhibitors, there was rapid excision. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment. Chapters 2-6 describe the single-color DNA SBS studies. Chapter 2 provides essential background on the structure of DNA, the DNA polymerase reaction, and several key DNA sequencing technologies, with an emphasis on the design of nucleotide analogues for the DNA SBS approach. Chapter 3 delineates a one-color fluorescent DNA SBS method based on a set of nucleotide reversible terminators (NRTs) comprising two orthogonal cleavable linkers, one fluorescent dye and one anchor. Chapter 4 describes a one-color hybrid DNA sequencing approach using a set of dideoxynucleotide analogues bearing two orthogonal cleavable linkers, one fluorophore and one anchor as well as a set of unlabeled NRTs. By introducing a pH responsive fluorophore into the design of nucleotide analogues, Chapter 5 demonstrates a novel type of single-color DNA SBS method using a set of NRTs comprising one pH-responsive fluorescent dye or one non-responsive fluorescent dye tethered with one cleavable linker. Chapter 6 presents another option for the single-color DNA sequencing technique using a set of deoxynucleotide analogues comprising the above pH responsive or non-responsive dyes tethered with a cleavable linker, along with a set of unlabeled NRTs. The one-color SBS approaches have the potential for higher sensitivity, miniaturization and cost effectiveness compared with four-color SBS methods. Finally, Chapter 7 summarizes the SARS-CoV-2 antiviral drug discovery and one-color sequencing techniques and discusses potential follow-up research on these projects.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA Polymerase II (RNAPII) is responsible for transcription of messenger RNA (mRNA) and many small non-coding RNAs. Progression through the RNAPII transcription cycle is orchestrated by combinatorial posttranslational modifications of the C-terminal domain (CTD) of the largest subunit of RNAPII, Rpb1, consisting of the repetitive sequence (Y1S2P3T4S5P6S7)n. Disruptions of proteins that control CTD phosphorylation, including the phosphatase Rtr1, cause defects in gene expression and transcription termination. There are two described RNAPII termination mechanisms. Most mRNAs are terminated by the polyadenylation-dependent cleavage and polyadenylation complex. Most short noncoding RNAs are terminated by the Nrd1 complex. Nrd1-dependent termination is coupled to RNA 3' end processing and/or degradation by Rrp6, a nuclear specific subunit of the exosome. The Rrp6-containing form a 3'-5' exonuclease complex that regulates diverse aspects of nuclear RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs). It remains unclear whether Rrp6 is directly involved in termination. We discovered that deletion of RRP6 promotes extension of multiple Nrd1-dependent transcripts resulting from improperly processed 3' RNA ends and faulty transcript termination at specific target genes. Defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of Nrd1 depletion from the nucleus. Our data indicate Rrp6 acts with Nrd1 globally to promote transcription termination in addition to RNA processing and/or degradation. Furthermore, we found that deletion of the CTD phosphatase Rtr1 shortens the distance of transcription before Nrd1-dependent termination of specific regulatory antisense transcripts (ASTs), increases Nrd1 occupancy at these sites, and increases the interaction between Nrd1 and RNAPII. The RTR1/RRP6 double deletion phenocopies an RRP6 deletion, indicating that the regulation of ASTs by Rtr1 requires Rrp6 activity and the Nrd1 termination pathway.

Chloroplasten höherer Pflanzen haben kleine Genome. Trotzdem ist ihre Transkriptionsmaschinerie sehr komplex. Plastidäre Gene werden von plastidenkodierten (PEP) und kernkodierten RNA-Polymerasen (NEP) transkribiert. In der vorliegenden Arbeit wurden Promotoren plastidärer Gene und Operons von Arabidopsis thaliana charakterisiert. Zur Unterscheidung zwischen NEP- und PEP-Promotoren wurden erstmals spectinomycinbehandelte, chlorophylldefiziente Arabidopsis-Pflanzen mit fehlender PEP-Aktivität verwendet. Obwohl für einige Gene auch einzelne Promotoren lokalisiert wurden, wird die Transkription der meisten plastidären Gene und Operons an multiplen Promotoren initiiert. Der Vergleich plastidärer Promotoren von Tabak und Arabidopsis zeigte eine hohe Vielfältigkeit der Promotornutzung, die möglicherweise auch in anderen höheren Pflanzen vorkommt. Dabei stellt die individuelle Promotornutzung eine speziesspezifische Kontrollmöglichkeit der plastidären Genexpression dar. Das Kerngenom von Arabidopsis beinhaltet zwei Kandidatengene der NEP, RpoTp und RpoTmp, welche Phagentyp-RNA-Polymerasen kodieren. In der vorliegenden Arbeit wurde die Wirkung veränderter RpoTp-Aktivität auf die Nutzung von NEP- und PEP-Promotoren in transgenen Arabidopsis-Pflanzen mit verminderter und fehlender RpoTp-Aktivität untersucht. Im Keimlingsstadium konnten Unterschiede in der Promotornutzung zwischen Wildtyp und Mutanten beobachtet werden. Fast alle NEP-Promotoren wurden in Pflanzen mit verringerter oder fehlender RpoTp-Aktivität genutzt. Dabei zeigten nur einige von ihnen eine geringere Aktivität, andere wiederum waren sogar verstärkt aktiv. Der starke NEP-Promotor des essentiellen ycf1 Gens wurde in jungen Keimlingen ohne funktionelle RpoTp nicht genutzt. Die Ergebnisse zeigen, dass NEP gemeinsam von beiden Phagentyp-RNA-Polymerasen RpoTp und RpoTmp repräsentiert wird und dass beide sowohl eine überlappende, als auch eine spezifische Rolle in der Transkription plastidärer Gene innehaben. / Although chloroplasts of higher plants have small genomes, their transcription machinery is very complex. Plastid genes of higher plants are transcribed by the plastid-encoded plastid RNA polymerase PEP and the nuclear-encoded plastid RNA polymerases NEP. Here, promoters of plastid genes and operons have been characterized in Arabidopsis thaliana. For the first time spectinomycin-treated, chlorophyll-deficient Arabidopsis plants lacking PEP activity have been used to discriminate between NEP and PEP promoters. Although there are plastid genes that are transcribed from a single promoter, the transcription of plastid genes and operons by multiple promoters seems to be a common feature. Comparison of plastid promoters from tobacco and Arabidopsis revealed a high diversity, which my also apply to other plants. The diversity in individual promoter usage in different plants suggests that there are species-specific solutions for attaining control over gene expression in plastids. The nuclear genome of Arabidopsis contains two candidate genes for NEP transcription activity, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. In this study the usage of NEP and PEP promoters has been analysed in transgenic Arabidopsis plants with reduced and lacking RpoTp activity. Differences in promoter usage between wild type and mutant plants were most obvious early in development. Nearly all NEP promoters were active in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene was not transcribed in young seedlings without functional RpoTp. These results provide evidence for NEP being represented by two phage-type RNA polymerases RpoTp and RpoTmp that have overlapping as well as specific functions in the transcription of plastid genes.

Licht und Pflanzenhormone sind essentiell für das Wachstum und die Entwicklung von Pflanzen. Es ist nur wenig darüber bekannt, wie sie die Transkription organellärer Gene beeinflussen. In Arabidopsis thaliana gibt es drei kernkodierte Phagentyp-RNA-Polymerasen (RpoT), welche für die organelläre Transkription verantwortlich sind. Diese werden in die Plastiden (RpoTp), die Mitochondrien (RpoTm) oder zu beiden Organellen (RpoTmp) transportiert. Neben den beiden kernkodierten RNA-Polymerasen (NEP) existiert in den Plastiden eine plastidärkodierte RNA-Polymerase (PEP), welche zusätzliche Sigmafaktoren zur Promotererkennung benötigt. Um die Lichtabhängigkeit der Expression der RpoT Gene sowie NEP-transkribierter Chloroplastengene zu analysieren, wurde die Akkumulation von RpoT- und rpoB-Transkripten in 7-Tage alten Keimlingen unter verschiedenen Lichtbedingungen mittels quantitativer real-time PCR untersucht. Die Änderungen in der Transkriptakkumulation deuten darauf hin, dass rote, blaue und grüne Wellenlängen die Expression der drei RpoT Gene unterschiedlich stark stimulieren. Untersuchungen an verschiedenen Lichtrezeptor-Mutanten zeigten, dass die Lichtinduktion der RpoT Genexpression überaus komplex ist und ein interagierendes Netzwerk aus multiplen Rezeptoren und Transkriptionsfaktoren an der Signalweiterleitung beteiligt ist. Das Phytohormon Cytokinin wird durch Histidin Kinase Rezeptoren (AHK) detektiert. Es gibt drei unterschiedliche Rezeptoren: AHK2, AHK3 und AHK4. Diese sind Teil eines Zwei-Komponenten-Systems, welches Signale mit Hilfe einer Phosphorylierungskette überträgt. Der Einfluss von Cytokinin auf die plastidäre Transkription wurde mit Hilfe von Cytokininrezeptor-Mutanten untersucht, um die Funktion von AHK2, AHK3 und AHK4 zu analysieren. Um weitere Informationen darüber zu erhalten, wie die plastidäre Transkription durch PEP mittels Cytokinin reguliert wird, wurden die Hormoneffekte auf die plastidäre Transkription in Sigmafaktor-Mutanten untersucht. / Light and plant hormones are essential for plant growth and development. Only little information is available about how these signals influence the transcription of organellar genes. Arabidopsis thaliana possesses three nuclear-encoded phage-type RNA polymerases (RpoT) for organellar transcription. They are imported into plastids (RpoTp), mitochondria (RpoTm), or into both organelles (RpoTmp). Besides the two nuclear-encoded plastid polymerases (NEP), plastids contain an additional plastid-encoded RNA polymerase (PEP), which needs additional sigma factors for promoter recognition. Interested in the expression of RpoT genes and NEP-transcribed plastid genes in response to light we analyzed transcript levels of RpoT and rpoB genes in 7-day-old wild-type plants under different light conditions by quantitative real-time-PCR. The observed changes in transcript accumulation indicated that red, blue, and green light differentially stimulated the expression of all three RpoT genes. Further analyses using different photoreceptor mutants showed that light induction of RpoT gene expression is surprisingly complex based on a network of multiple photoreceptors an d downstream pathways. Cytokinin signals are perceived by the histidine kinase (AHK) receptor family. There exist three different membrane-bound receptors: AHK2, AHK3 and AHK4/CRE1. These receptors are part of a two-component signaling system which transfers signals via phosphorelay mechanisms. Interested in the potential role of AHK2, AHK3 and AHK4/CRE1 in the transduction of cytokinin signals into the chloroplast, we analyzed the influence of cytokinin on plastidial transcription in receptor mutants. To gain more information on how plastid transcription by PEP is regulated by cytokinin, the influence of cytokinin in sigma factor mutants was also studied.

Le virus de l'hépatite C (HCV) est un virus à ARN qui synthétise ses nouveaux génomes dans les cellules hôtes infectées grâce à une ARN polymérase ARN dépendante (RdRp), appelée NS5B. Cette polymérase a été pendant longtemps une cible majeure dans la recherche d'antiviraux contre l'hépatite C. Aujourd'hui, de nombreux antiviraux ont été approuvés dans le traitement de l’hépatite C ciblant différentes protéines virales, entre autre, NS5B. Le sofosbuvir qui cible le site actif de NS5B est un antiviral qui a démontré une efficacité extraordinaire dans le traitement anti-HCV.Durant ces dernières années, les recherches sur NS5B ont permis de caractériser cette protéine, à la fois structuralement et biochimiquement. La richesse des connaissances ainsi accumulées fait de NS5B un excellent modèle pour les RdRp des virus à ARN.Toutefois, peu d'études portent sur le mécanisme d’acheminement et de sélection des ribonucléotides au site actif de NS5B, et de façon générale, sur la description atomique du mécanisme de réplication assuré par NS5B, qui implique deux dications magnésium pour la catalyse comme chez toutes les polymérases de cette famille.Durant ce travail, nous avons exploité les données structurales issues de complexes ternaires (NS5B+RNA+nucleotide) obtenus en 2015 par cristallographie à l'échelle atomique. Nous avons utilisé ces structures pour mener des études de modélisation moléculaire, essentiellement par dynamique moléculaire afin d’aborder la question de l'acheminement des nucléotides vers le site actif de NS5B.Nous avons eu recours à la fois à des méthodes de dynamiques moléculaires classiques et des méthodes de dynamiques moléculaires dites biaisées telles que différentes méthodes de dynamiques moléculaires dirigées (SMD et TMD) et la dynamique moléculaire accélérée (aMD).Nos résultats indiquent que l’acheminement du nucléotide au site actif associé à un magnésium Mg(B) est contrôlé tout au long du tunnel par différents éléments de NS5B. Initialement, le nucléotide se lie à une région proche de la boucle qui surplombe le tunnel lui permettant, grâce aux interactions qu’il établit avec sa partie triphosphate, de s’orienter base en avant. Ensuite, le nucléotide atteint un point de contrôle formé essentiellement par le motif F3 (R158) et le motif F1(E143). Le nucléotide reste lié à ce site jusqu’à l’arrivée du second dication magnésium (Mg(A)) qui provoque des réarrangements structuraux, notamment au niveau du motif F3, entrainant l’avancée du nucléotide vers le site actif. Une fois ce point de contrôle passé, le nucléotide interroge alors la base du brin d’ADN matrice sans s’insérer entièrement dans le site actif.Nos simulations ont clairement établi que les dernières étapes d’entrée du nucléotide sont finement régulées par l’arrivée du second dication magnésium qui a donc un rôle de coordinateur en plus de son rôle connu dans la catalyse.Ce mécanisme d’entrée semble être spécifique aux RdRp virales et permet de comprendre pourquoi les analogues de nucléotides peuvent être aussi efficaces contre les virus à ARN. / The hepatitis C virus is an RNA virus that synthesises its new genomes in the infected host cells thanks to an RNA-dependent RNA polymerase (RdRp) termed NS5B. This polymerase has been a prime target for antiviral therapy. Numerous direct antiviral drugs are now approved in the HCV treatment and allow very high rates of treatment success. These drugs target among others the HCV NS5B RdRp with the sofosbuvir being one of the most successful drugs.Tremendous efforts have been made in the past decades to characterize NS5B, in particular structurally and biochemically. However, there is little information about the molecular mechanisms of NS5B ribonucleotides entry and selection and in general on the atomistic details of the RNA replication mechanism, although the involvement of two magnesium dications in catalysis is well established in this family of polymerases. Since 2015, structures of ternary complexes of NS5B have been resolved by crystallography offering very valuable details about the binding of nucleotides at the NS5B active site.In this work, we took advantage of these structural data to address the ribonucleotides entry and to further explore the nucleotide addition cycle in NS5B using molecular modelling and molecular dynamics simulations. We used both conventional molecular dynamics techniques and biased simulations that enhance sampling such as Steered Molecular Dynamics (SMD), Targeted Molecular Dynamics (TMD) or accelerated Molecular dynamics (aMD).Based on our modelling results, we found that the access to the active site through the nucleotides tunnel is checked by successive NS5B elements. First, the entering ribonucleotide together with an associated magnesium Mg(B) binds next to a loop that overhangs the nucleotide tunnel and interactions with its triphosphate moiety orient it base-first towards the active site. Second, the ribonucleotide encounters a checkpoint constituted by the residues of motif F3(R158) and motif F1(E143) where it is blocked until the arrival of a second magnesium ion, the Mg(A). This allowed the motif F3 to undergo small structural rearrangements leading to the advancement of the nucleotide towards the active site to interrogate the RNA template base prior to the complete nucleotide insertion into the active site.Our simulations pointed out that these dynamics are finely regulated by the second magnesium dication, thus coordinating the entry of the correct magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. This entry mechanism is specific to viral RdRps and may explain why modified ribonucleotides can be so successful as drugs against RNA viruses.

Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcerative disease that facilitates the transmission of HIV-1. H. ducreyi also causes non-sexually transmitted cutaneous ulcers in children in tropical regions. During human infection, H. ducreyi is subject to a variety of stresses. The stringent response is a bacterial stress response system induced by nutrient limiting conditions and mediated by guanosine tetra- and pentaphosphate [(p)ppGpp] and the transcriptional regulator DksA. (p)ppGpp and DksA jointly interact with RNA polymerase to regulate genes critical for bacterial survival. We hypothesized that the stringent response is required for H. ducreyi virulence in humans. A ΔrelAΔspoT mutant, which is unable to synthesize (p)ppGpp, was partially attenuated for abscess formation in human volunteers. Loss of (p)ppGpp increased bacterial resistance to phagocytosis and stationary phase survival; however, the mutant was more sensitive to oxidative stress. A ΔdksA mutant was also partially attenuated in humans. The ΔdksA mutant behaved like the (p)ppGpp mutant in stationary phase survival and sensitivity to oxidative stress, but exhibited decreased resistance to phagocytosis. Both mutants had decreased adherence to fibroblasts, but the mechanisms underlying the adherence defect were distinct. To better understand the roles of (p)ppGpp and DksA in regulating gene expression, we performed transcriptome analysis of the parent and mutant strains. (p)ppGpp and DksA deficiency resulted in dysregulation of multiple genes including several known virulence determinants. At stationary phase, (p)ppGpp and DksA targets were not identical but significantly overlapped; as the mutants were phenotypically distinct, this finding underscores both the unique and joint roles DksA and (p)ppGpp play in regulation of H. ducreyi virulence. We conclude that (p)ppGpp and DksA play significant roles in H. ducreyi pathogenesis. This is the first study to show that the stringent response has a direct role in the ability of a bacterial pathogen to cause disease in humans.