Escherichia coli transcription termination factor Rho is a hexameric RNA/DNA helicase that terminates transcription using energy derived from the hydrolysis of ATP. The ATP binding sites of Rho are located at the interfaces of adjoining subunit Cterminal domains and have the Walker A and B motifs, characteristic of many ATPases (Skordalakes & Berger, 2003; Richardson 2002). Available Rho crystal structures capture the protein with its active site in an open configuration that must close to permit ATP hydrolysis. Because of this, the identities of active site residues predicted to mediate ATP hydrolysis are uncertain. To determine which amino acids activate water, stabilize transition state, sense the γ- phosphoryl group, and coordinate the magnesium ion of MgATP, we have carried out site-specific mutagenesis on candidate residues which are conserved across bacterial species, and characterized the relevant properties of the mutant proteins. The residues chosen were E211 as the water activator, R212 as the γ sensor, R366 as the arginine finger, and D265 as the residue that coordinates Mg2+. Each mutant protein was investigated for its ability to oligomerize as hexamers, assayed for ATPase activity, ATP and RNA binding, and pre-steady-state kinetics. The results show that the mutant proteins form hexamers similarly as to wild type Rho. The RhoE211 mutants display at least a 200-fold lower activity as ATPases, bind both ATP and RNA with similar affinities as the wild type protein, and display no burst in pre-steady-state kinetics. RhoR212A protein has 20-fold lower activity as an ATPase compared to wild type Rho, binds ATP with at least a 50-fold weaker affinity, and RNA with a 2-fold higher KD compared to wild type Rho. RhoR366A functions as an ATPase with 50-fold lower activity, binds RNA with similar affinity as wild type Rho and binds ATP with a 5- fold weaker affinity. RhoD265N displays 150-fold lower ATPase activity compared to the wild type enzyme, binds ATP with a 10-fold weaker affinity, and binds RNA with similar affinity as wild type Rho. Pre-steady-state kinetics studies indicate that the mutant proteins investigated show no burst kinetics, indicating a failure or a significantly slower rate of the hydrolysis (chemistry) step. It is possible that the rate-limiting step is the chemistry step in these mutant proteins, contrary to the wild type protein where the chemistry step is much faster (300/s). Together, the results obtained are consistent with the proposed roles for these residues: E211 is involved in activating a water molecule, R212 functions as the γ sensor, R366 functions as the arginine finger and D265 is involved in coordination of the Mg2+ ion. This study has elucidated the mechanism of ATP hydrolysis, by determining some of the key residues involved in the hydrolysis reaction. This study is only a part of the characterization of the active site residues. There might be other residues involved in one or all of the functions proposed. Utilizing the findings from this study, other experiments and models can be implemented to understand how Rho hydrolyzes ATP and utilizes the energy to move along the RNA molecule and functions as a helicase. / Biochemistry

L’expression des gènes est un ensemble hautement régulé de mécanismes ayant pour objectifs de synthétiser les protéines fonctionnelles dont la cellule a besoin à partir des codes inscrits dans l’ADN. Pour contrôler la quantité de signal utilisé et pour que ce message puisse physiquement traverser la cellule, celle-ci utilise la transcription des ARN messagers comme intermédiaire. Pour assurer la qualité de ce signal et pour contrôler son niveau d’expression, plusieurs mécanismes de dégradation des ARNm se coordonnent dans les organismes en fonction de leurs spécificités propres. La littérature a depuis longtemps démontré les liens entre les machineries de synthèse des ARNm et celles de leur dégradation en identifiant comment ceux-ci travaillent ensemble pour assurer une bonne régulation génique. Un de ces mécanismes induit une terminaison dans la région non-codante en 3’ de certains gènes à partir d’un clivage par la ribonucléase III. Dans ce mémoire, nous voulons démontrer qu’un mécanisme similaire dépendant de la ribonucléase III peut induire une terminaison prémature de la transcription à l’intérieur même de séquences codantes. Ce mécanisme semble être indépendant du promoteur et du terminateur des gènes, préférant réguler sa sélectivité à partir de la structure liée au clivage de l’ARNm. Plusieurs séquences et structures sous forme de tige-boucles d’ARNm peuvent être reconnues par la ribonucléase III. Cependant, il existe des différences fonctionnelles entre les différentes tige-boucles et toutes n’induisent pas un mécanisme de terminaison prématurée. Comme ce type de mécanisme doit être inductible et/ou permissible afin de ne pas empêcher complètement l’expression des gènes cibles, nous pouvons potentiellement faire affaire à un nouveau modèle de régulation génique. / Abstract : Gene expression is a highly regulated coordination of processes with the objective of producing functional proteins from the DNA code of the cell. To control the amount of proteins produced, cells use messenger RNAs as an intermediate to permit genomic information to move across the cell. To assure the quality of this signal and to control the level of gene expression, many RNA degradation mechanisms coordinate together according to their own specificities. Scientific literature has demonstrated long ago the existing interactions between RNA synthesis and RNA degradation pathways and how they closely work together to achieve viable gene expression regulation. One of those mechanisms induces transcription termination in the 3’ untranscribed region of coding genes initiated with a RNA cleavage from a ribonuclease III. In this master thesis, we show that a similar RNase III dependent mechanism can induce premature transcription termination inside the coding sequence. This mechanism seems promotor and terminator independent and depends mostly on the sequence coding for a stem-loop structure in the mRNA. Different sequences can induce a stem-loop structure recognizable by RNase III. However, there are some functional differences between stem-loop structures and not all of them can induce premature transcription termination. Since this mechanism must not happen every time and somehow must be inducible to permit gene expression when needed, this could possibly lead to a new gene regulation model.

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.

The CCCTC-binding factor (CTCF) is a versatile, multifunctional zinc-finger protein involved in a broad spectrum of cellular functions. In mammalian cells, CTCF functions together with the Cohesin complex, an essential regulator of sister chromatid cohesion. Together, CTCF and Cohesin have been shown to regulate gene expression at a genome-wide level in mammalian cells. In the yeast Saccharomyces pombe, Cohesin has been implicated in transcription termination of convergently transcribed genes, in a cell cycle dependent manner. The aim of this thesis was to investigate the possibility of direct transcriptional involvement of CTCF and Cohesin in human cells. The first model system applied for this experimental purpose was the β-globin gene with introduced canonical CTCF-binding sites replacing the endogenous Co- Transcriptional Cleavage (CoTC) element downstream of β-globin. The results obtained indicate that recruitment of CTCF to the β-globin 3` flanking region does not prevent read-through transcription. However, CTCF-binding does mediate RNA Polymerase II (Pol II) pausing at the site of recruited CTCF. This results in more efficient pre-mRNA 3` end processing and therefore rescues β-globin mRNA to wild type levels. Cohesin was not detected at the introduced CTCF-binding sites. These results are a contribution to our understanding of the spatio-temporal requirements for cotranscriptional events like 3` end pre-mRNA processing and Pol II kinetics. The second part of my thesis presents an investigation on the involvement of CTCF and Cohesin in lipopolysaccharide (LPS)-induced Tumor Necrosis Factor α (TNFα) gene expression regulation in human monocytes and differentiated M1- and M2-type macrophages. These studies provide first evidence of Cohesin recruitment to the TNFα gene body and its regulatory NFκB-binding sites. Differences in the recruitment profiles obtained indicate potential regulatory differences of TNFα among the three cell types. Preliminary data provide an insight into the effects on TNFα mRNA levels upon down-regulation of Cohesin subunits.

L’ARN polymérase II (ARNPII) synthétise des ARNs codants et des ARNs non-codants (ARNnc) tels que les petits ARNs nucléaire/nucléolaire (sn/snoRNAs) et les CUTs (Cryptic Unstable Transcripts). Les CUTs sont des transcrits ubiquitaire souvent produits dans des régions codants dont la transcription peut interférer avec l’expression des gènes. Le contrôle de l’expression des ARNnc est essentiel et se fait aux niveaux de la terminaison de la transcription et la dégradation de l’ARN. Chez la levure Saccharomyces cerevisiae la terminaison de la transcription des gènes codants est effectuée par le Facteur de Clivage et de Polyadénylation (CPF), tandis que les ARNnc courts sont terminés par le complexe Nrd1p-Nab3p-Sen1p (NNS). La terminaison de la transcription est régulée par la phosphorylation du domaine C-terminal (CTD) de l’ARNPII composé de répétitions du motif Y1S2P3T4S5P6S7. Un niveau élevé de phosphorylation des résidus Ser5 près du promoteur permet l’activité du complexe NNS. La phosphorylation des résidus Ser2 est catalysée durant la transcription par la kinase Ctk1p et ces résidus sont reconnus par des éléments de la voie CPF. Mon travail de thèse a porté sur le mécanisme de terminaison de la transcription par le complexe NNS. La terminaison NNS dépend de la liaison de Nrd1p et Nab3p à des motifs dans l’ARN naissant et l’activité hélicase de Sen1p qui provoque le relarguage de la polymérase. La sous-unité Nrd1p interagit avec le domaine CTD de l’ARNPII phosphorylé sur Ser5 à travers son domaine CID (CTD-interaction domain). Le rôle du CID dans la terminaison à été proposé mais pas encore clairement démontré. En collaboration avec le groupe de P. Cramer (Université Louis-et-Maximilien de Munich Allemagne) nous avons mis en évidence que le CID est requis pour une terminaison efficace par la voie NNS et qu’il est important pour le recrutement de Nrd1p sur l’ARNPII. Le CID est aussi impliqué de manière directe ou indirecte dans l’interaction de Sen1p avec Nrd1p et Nab3p. En parallèle, avec le groupe de F. Holstege (Université Centre Médicale de Utrecht, Pays-Bas) nous avons montré que la phosphorylation en Ser2 du domaine CTD est requise pour une terminaison efficace par la voie NNS. De manière surprenante, ce résidu joue un rôle mineur dans la terminaison des ARNs codants effectuée par le complexe CPF. Les ARNs naissant terminés par le complexe NNS sont rapidement ciblés par le complexe nucléase exosome/Rrp6p et son cofacteur TRAMP ce qui mène a la maturation des sn/snoRNAs et la destruction des CUTs. Le complexe NNS interagit in vivo avec l’exosome et le complexe TRAMP, ce qui facilite la dégradation. Cependant les détails moléculaires de cette interaction restent inconnus. Nous avons montré que le domaine CID est requis et suffisant in vivo et in vitro pour l’interaction de Nrd1p avec la partie C-terminale de la sous-unité Trf4p du complexe TRAMP, que nous avons appelé NIM (Nrd1p-Interaction Motif). En collaboration avec le groupe de R. Stefl (Université Masaryk, République Tchèque) nous avons étudié par spectroscopie RMN la structure de ce motif NIM lié au CID. Nous avons mis en évidence que le CID lie le NIM et le CTD de façon similaire, et que ces interactions sont mutuellement exclusives. Le NIM se lie au CID environ 100 fois plus fortement qu’au CTD. Nous proposons que ces interactions alternatives de Nrd1p définissent des formes différentes du complexe NNS, une qui fonctionne dans la terminaison de la transcription, l’autre qui est active dans la dégradation. In vitro l’interaction du NIM avec le CID stimule l’activité poly(A)-polymérase de Trf4p ce qui suggère une fonction importante de cette interaction dans la dégradation. Nous montrons aussi que Rrp6p interagit directement avec Trf4p et cette liaison in vivo sert à recruter le complexe TRAMP à l’exosome Nous proposons que ce jeu serré d’interactions entre les complexes NNS, TRAMP et l’exosome/Rrp6p contribue à augmenter l’efficacité de dégradation de l’ARN in vivo / The RNA polymerase II (RNAPII) synthesizes protein-coding RNAs and many non-coding RNAs (ncRNAs) such as small nuclear/nucleolar (sn-/snoRNAs) and Cryptic Unstable Transcripts (CUTs). CUTs are ubiquitously transcribed including overlapping and antisense to genes, which can interfere with gene expression. Control of ncRNA expression is vital and also operates at the level of transcription termination and RNA degradation.In yeast Saccharomyces cerevisiae transcription of protein-coding genes is terminated by the Cleavage and Polyadenylation Factor (CPF), while short ncRNAs are generated by transcription termination dependent from the Nrd1p-Nab3p-Sen1p (NNS) complex. Transcription termination is regulated by phosphorylation of the carboxy-terminal domain (CTD) of the Rpb1p subunit of RNAPII, composed of repeats of the Y1S2P3T4S5P6S7 motif. Promoter-proximal high levels of serine 5 phosphorylated (Ser5P) CTD favors the function of the NNS pathway while the Ser2 phosphorylated mark (Ser2P), which is gradually introduced during transcription by Ctk1p, is recognized by components of the CPF pathway. The study of the mechanism of action of the NNS complex was the subject of my PhD work.NNS-dependent transcription termination is driven by the recognition of four nucleotide motifs in the nascent RNA by Nrd1p and Nab3p and the release of the RNAPII by the Sen1p helicase. Nrd1p interacts with the CTD-Ser5P via its CTD-interaction domain (CID). Thus a role of the CID in termination was anticipated but not demonstrated. In collaboration with the group of P. Cramer (Ludwig Maximilian University of Munich, Germany), we have shown that the Nrd1p CID domain is required for efficient transcription termination at most NNS-target genes and that it is important for the recruitment of Nrd1p to the RNAPII. This domain is also involved, directly or indirectly, in the interaction of the Sen1p helicase with Nrd1p and Nab3p. In the second project, in collaboration with F. Holstege group (University Medical Center Utrecht, Netherlands), we have shown that the CTD-Ser2P mark is important for efficient transcription termination by the NNS pathway but, surprisingly, it appears to play a minor role in termination of mRNA-coding genes by the CPF-complex.Shortly after NNS-dependent termination, the released ncRNAs are targeted by the nuclear exosome/Rrp6p nuclease complex and its cofactor the TRAMP which results in trimming of sn-/snoRNAs to a mature form and complete degradation of CUTs. The NNS complex co-purifies in vivo with the TRAMP/exosome, which is believed to facilitate subsequent degradation and processing. However, the molecular details of this interaction are unknown. We show that the CID is required and sufficient in vivo and in vitro for the interaction of Nrd1p with a motif present in the C-terminal region of Trf4p, which we called NIM (for Nrd1p-Interaction Motif). In collaboration with the group of R. Stefl (Masaryk University, Czech Republic), we obtained the NMR structure of the CID bound to the NIM and demonstrated that the CID binds in a similar manner to the CTD and the NIM. The CID interacts with the CTD and the NIM in a mutually exclusive manner and the former interaction is roughly 100 times stronger than the first. We propose that these alternative interactions represent two forms of the NNS complex, one functioning in termination and the other in degradation. Importantly, the NIM-CID interaction is likely to be functionally relevant since in vitro it results in the stimulation of the polyA polymerase activity of the Trf4p. We further show that Trf4p interacts directly with Rrp6p, which in vivo serves to recruit the TRAMP to the core exosome complex. This tight interplay between the NNS, TRAMP and exosome/Rrp6p complexes most likely accounts for the efficiency of RNA degradation in vivo.

No description available.

Chez les eucaryotes, la maturation de l’extrémité 3’ des ARNs messagers a lieu lors de la transcription et regroupe deux étapes : le clivage endonucléolytique du transcrit au niveau d’un site spécifique et l’ajout d’une queue poly(A) sur le fragment en amont du site de clivage. Chez S. cerevisiae, le complexe de polyadénylation est formé par 20 protéines, regroupées principalement en deux sous-complexes : CF IA et CPF. Nous nous intéressons plus spécifiquement à Pcf11, sous-unité du complexe CF IA. Pcf11 est formé de sept sous-domaines, mais la fonction d’une grande partie de la protéine n’est pour l’instant pas connue. Par exemple, aucune fonction n’est associée à la région située entre le domaine d’interaction avec le CTD de l’ARN polymérase II (CID) et une répétion de 20 résidus glutamines. Récemment, la structure de ce domaine, appelé 2nd NTD a été décrite. Pour essayer de comprendre la fonction du 2nd NTD et des motifs liant le zinc encadrant le domaine d’interaction avec Clp1, nous avons mis en place une stratégie systématique de mutagénèse, soit par délétions, soit par mutations ponctuelles. Le 2nd NTD est formé de trois hélices α et interagit avec l’ARN. La délétion de ce domaine conduit à un phénotype de croissance lente chez la levure et un défaut de terminaison de transcription des snoRNAs. Malgré une similarité de structure et de fonction, le 2nd NTD présenterait une fonction indépendante. La fonction des motifs liant le zinc n’est pour l’instant pas connue. Cependant, la mutation de l’un de ces deux motifs conduit à un défaut de clivage et de polyadénylation in vitro. La mutation des deux motifs est létale chez la levure. / In eukaryotes, poly (A) tails are added to nuclear pre-mRNA 3'-ends in the two steps of cleavage and polyadenylation. This co-transcriptional processing requires the activity of a large protein complex comprising at least 20 different polypeptides in yeast organized primarily into the two factors CF IA and CPF. We are interested in the functional characterization of Pcf11, a CF IA subunit. The Pcf11 protein is organized into seven different domains, but here is still a large portion of the polypeptide that has not yet been characterized. For example the region from the end of the CTD interaction domain (CID) to an uninterrupted stretch of 20 glutamine residues has no known function. Recently, the structure of this region, called the 2nd NTD have been characterized. To gain insight into the function of the 2nd NTD and the two zinc fingers motif surrounding the Clp1 interaction domain, we have employed a systematic strategy of mutagenesis, either by deletion or via point mutations. The 2nd NTD is a folded domain composed of three α-helices. The deletion of this domain induced a severe defect of growth in yeast and impaired transcription termination of snoRNAs. Despite its similarity in structure and function with the CID, the 2nd NTD seems to act like an independent RNA binding domain. We don’t know yet the real function of the two zinc fingers motif at the C-terminal region of Pcf11, but the mutation of Cystein residues into serine of one of the two motifs impaired cleavage and polyadenylation. The mutation of the first motif is less harmful than the mutation of the second motif. The simultaneous mutation is lethal in yeast.

Two mechanisms, intrinsic and factor-dependent, have evolved for accomplishing the termination of transcription in eubacteria. In this thesis, the first chapter is an introduction to the topic that presents what is known about the mechanisms of termination. The properties of the primary and secondary ‘players’- intrinsic terminators, Rho protein, rho-dependent terminators, RNA polymerse and Nus factors - are presented and the known mechanisms by which termination functions are discussed. In Chapter 2, a detailed analysis of intrinsic terminators – their differential distribution, similarity and divergence - has been penned. The database, compiled using the program GeSTer (Genome Scanner for Terminators), comprises ~2000 sequences and is one of the largest of its kind. Furthermore, analyzing the data from over 700 bacteria reveals how different species have fine-tuned intrinsic terminators to suit their cellular needs. Non-canonical intrinsic terminators emerge to be a significant fraction of the observed structures. The conserved structural features of identified intrinsic terminators are discussed and the relationship between the two modes of termination is assessed. Chapter 3 deals with the importance of transcription termination in regulating horizontally acquired DNA. The results show that genomic islands are scarce in intrinsic terminators and thus constitute most likely sites for Rho-dependent termination. Plausible reasons for why such a scenario has evolved are discussed and a generally applicable model is presented. Chapters 4 and 5 focus on Rho protein from Mycobacterium tuberculosis. In silico identification of M. tuberculosisgenes that rely on MtbRho-dependent termination is followed by experimental validation. The data show that Rho-dependent termination is the predominant mechanism in this species.MtbRho is a majorly expressed protein that governs termination of protein-coding and non-protein coding genes. Further, MtbRho can productively interact with RNA that has considerable secondary structure. Such interactions cause conformational changes in the enzyme. Given that MtbRho has to function with a GC-rich transcriptome, the altered properties could have evolved for optimal function. Taken together, the thesis extends our current understanding of both modes of termination. The importance of non-canonical intrinsic terminators in mycobacteria and other organisms is discussed. The unusual function of Rho and its predominant role in mycobacteria is elucidated. Finally, the inter-relationship between the two modes of termination is also discussed.