Regensburg, Univ., Diss., 2008

No description available.

Dans les cellules eucaryotes, la transcription de l’ADN nucléaire est effectuée grâce à trois ARN Polymérases ADN dépendantes (Pol). La Pol I transcrit les ARN ribosomaux, la Pol II produit essentiellement des ARN messagers et des micro ARN alors que la Pol III transcrit des petits ARN non traduits impliqués dans une variété de processus cellulaires essentiels tels que la traduction, l’épissage ou la régulation de la transcription. L’ARN Polymérase III humaine est un complexe enzymatique constitué de 17 sous-unités dont la plupart sont apparentées à des sous-unités de la Pol I et/ou la Pol II. Une de ces sous-unités, hRPC32 est présente sous forme de deux paralogues α et β codés par deux gènes différents où hRPC32β est exprimée de façon ubiquitaire alors que hRPC32α est exprimée spécifiquement dans les cellules transformées ou non différenciées. Au sein de la Pol III, hRPC32α/β, hRPC62 et hRPC39 forment un sous-complexe ternaire stable dissociable de l’enzyme. Ces trois sous-unités sont spécifiques à la Pol III et sont impliquées dans l’étape d’initiation de la transcription. L’objectif de ce travail de thèse est d’éclaircir les mécanismes de fonctionnement du sous-complexe hRPC62-hRPC39-hRPC32α/β. Le travail réalisé a permis, dans un premier temps, de cartographier les domaines d’interaction entre la sous-unité hRPC62 et les deux paralogues α et β de la sous-unité hRPC32. Ensuite, nous avons mené une analyse biochimique des activités enzymatiques des protéines recombinantes de hRPC62 et hRPC39. Cette analyse a montré que hRPC62 possède des homologies fonctionnelles avec TFIIEα, un facteur de transcription de l’ARN Polymérase II récemment décrit comme étant un homologue structural de la sous-unité hRPC62. Ces données supportent le modèle suggérant que certaines sous-unités des ARN Polymérases peuvent être considérées comme des facteurs de transcription recrutés de façon permanente à l’enzyme. / In eukaryotes, nuclear transcription is carried out by DNA dependent RNA polymerases (Pol) I, II and III. Pol I transcribes ribosomal RNA’s, Pol II produces essentially messenger and micro RNA’s whereas Pol III transcribes small untranslated RNA’s involved in a variety of cellular processes such as translation, splicing or the regulation of transcription. Human Pol III is a multi-subunit enzyme composed of 17 subunits. The majority of these subunits are homologous or closely related to Pol II and/or Pol I subunits. However, five subunits are specific to Pol III with no counterparts in Pol I or Pol II. One of the Pol III specific subunits, hRPC32 has two paralogues, α and β, expressed from two different genes. hRPC32β is expressed ubiquitously while hRPC32α expression is specific to transformed or non-differentiated cells. Within the Pol III enzyme, hRPC32α or β associate with two other Pol III specific subunits, hRPC62 and hRPC39, to form stable ternary sub-complexes thought to be implicated in transcription initiation. The purpose of this work was to clarify the functional mechanism of hRPC32α/β-hRPC62-hRPC39 sub-complexes. In this study, we first mapped the protein-protein interaction of hRPC62 with hRPC32α and hRPC32β. Second, we performed a biochemical study of hRPC62 and hRPC39 enzymatic activities. This analysis showed that hRPC62 has functional homologies with TFIIEα, a Pol II transcription factor recently described as a structural homolog of hRPC62. These results support the model that certain RNA polymerase subunits can be considered as transcription factors that have been stably recruited to the enzyme.

Le complexe condensine est le moteur de la condensation mitotique des chromosomes, un processus essentiel à la stabilité du génome au cours de la division cellulaire. De nombreuses données publiées indiquent qu’il existe des liens fonctionnels étroits entre le processus de transcription des gènes et le processus d’organisation des chromosomes par condensine. Ces données sont toutefois souvent contradictoires et aucun modèle ne fait actuellement consensus pour expliquer les liens entre transcription et condensine. Au cours de cette thèse, nous avons montré chez la levure Schizosaccharomyces pombe qu’en l’absence de l’hélicase à ADN/ARN Sen1, condensine s’accumule spécifiquement à proximité des gènes transcrits par l’ARN Polymérase III. Nous avons utilisé ces observations pour mieux comprendre les liens entre transcription par l’ARN polymérase III et le positionnement de condensine. Nos données montrent que Sen1 est un cofacteur de l’ARN Polymérase III impliqué dans la terminaison de la transcription. Ce résultat est important car il démontre que les modèles existants qui affirment que l’ARN polymérase III termine de transcrire de façon autonome sont erronés. Nous avons ensuite démontré que les défauts de terminaison de l’ARN polymérase III observés en l’absence de Sen1 suffisent entièrement à expliquer l’accumulation de condensine en ces sites. Cette observation importante démontre que le contrôle de qualité de la transcription est directement impliqué dans le positionnement de condensine sur les chromosomes en mitose. Nos résultats nous permettent de proposer qu’au-delà d’un certain seuil, la densité en ARN polymérases est un obstacle à la translocation de condensine sur les chromosomes. / The condensin complex is a key driver of chromosome condensation in mitosis. The condensin-dependent assembly of highly compacted chromosomes is essential for the faithful transmission of the genome during cell division. Many independent studies have established that gene transcription impacts the association of condensin with chromosomes, but the molecular mechanisms involved are still unclear. This is especially true as a number of sometimes contradictory mechanisms have been proposed so far. Here, we show in Schizosaccharomyces pombe that condensin accumulates specifically in the vicinity of a subset of RNA polymerase III-transcribed genes in the absence of the conserved DNA/RNA helicase Sen1. We demonstrate that Sen1 is a cofactor of RNA polymerase III (RNAPIII) required for efficient transcription termination. These results are important because they fundamentally challenge the pre-existing view that RNAPIII terminates transcription autonomously. Strikingly, we show that the RNAPIII transcription termination defects are directly responsible for the accumulation of condensin in the absence of Sen1. This indicates that the quality control of transcription impacts the distribution of condensin on mitotic chromosomes. We propose that above a certain density threshold, the accumulation of RNAPIII constitutes a barrier for the translocation of condensin on chromosomes.

7SK RNA is a highly abundant noncoding RNA in mammalian cells whose function in transcriptional regulation has only recently been elucidated. Despite its highly conserved sequence throughout vertebrates, all attempts to discover 7SK RNA homologues in invertebrate species have failed so far. Here we report on a combined experimental and computational survey that succeeded in discovering 7SK RNAs in most of the major deuterostome clades and in two protostome phyla: mollusks and annelids. Despite major efforts, no candidates were found in any of the many available ecdysozoan genomes, however. The additional sequence data confirm the evolutionary conservation and hence functional importance of the previously described 3´ and 5´ stemloop motifs, and provide evidence for a third, structurally well-conserved domain.

The work in the Brown laboratory has two long-range objectives. Both are derived from an interest in the replication of the genome of Gram-positive eubacteria. One objective is to gain a deeper understanding of the structure and function of DNA polymerase III, the unique species of DNA polymerase which is essential for chromosome replication. The second objective, the one from which this thesis is derived, is to determine whether a selective inhibitor of this DNA polymerase can serve as a basis for producing a new generation of clinically useful Gram-positive-selective antimicrobial agents. The polymerase III-specific inhibitor prototypes investigated in this work are members of a family of simple 6-substituted uracils. The following members of this family, TMAU and EMAU, were used as platforms for the manipulation of the N3 atom (arrow), the only ring component which could be substituted without severe reduction of inhibitory activity. The N3 position was substituted with a series of alkyl groups of increasing size. The resulting structure-activity relationships at the level of the polymerase was consistent with the presence of an N3-specific subdomain within the inhibitor binding site which could accommodate a wide variety of substituents. Although specific alkyl substituents at N3 also significantly enhanced the antibacterial potency of TMAU and EMAU, the respective compounds were found to have insufficient aqueous solubility for successful application in in vivo infection. To increase aqueous solubility, the N3 atom of the EMAU platform was substituted with selected hydroxy- and methoxyalkyl groups. The latter agents retained both anti-polymerase and antibacterial activity, and, as expected, they displayed a combination of lipid and aqueous solubility favorable to efficacy in in vivo infection. Two of the agents, N3-hydroxypropyl- and N3-methoxypropyl-EMAU were examined for their ability to protect mice from lethal staphylococcal infection. Both were found to be active in this model. In sum, the results of this work demonstrated, for the first time, that: (1) the eubacterial replication-specific DNA polymerase III is a valid target for antibiotic development, and (2) the N3-substituted 6-anilinouracil platform has strong potential as a basis for novel antibiotics useful against Gram-positive bacterial infection.

The major research interest of our laboratory is focused on the replication-specific DNA polymerase III (pol III) family in Gram+ bacteria, and has used Bacillus subtilis (BS) as the primary model enzyme for study. The long range objective of the work of the laboratory is to gain a deeper understanding of the structure and function of Gram+ bacterial DNA polymerase IIIs, a structurally unique class of DNA-dependent DNA polymerase which are uniquely susceptible to inhibition by a specific class of dGTP analogs. The project described in this thesis dissertation deals specifically with the pol III of the Gram+ organism Staphylococcus aureus, and involves the isolation and characterization of DNA pol III from this clinically relevant pathogenic bacterium. A homology-based strategy was devised to clone the structural gene specifying DNA polymerase III of Staphylococcus aureus, SA polC. SA polC was found to contain a 4305-bp open reading frame (ORF) encoding a 162.4 kDa polypeptide, and mapped between Ω1074[Tn551] and recA/ngr on the genome map of S. aureus NCTC 8325. The 1435 codon ORF was engineered into the E. coli expression plasmid pBS(KS) under the control of the lac promoter and its repressor. The translational signals of SA polC were reengineered using expression cassette PCR (ECPCR) to optimize the in vitro expression of SA polC in E. coli. Derepression of E. coli transformants carrying the recombinant vector generated high level expression of active recombinant pol III. The recombinant SA pol III was purified to greater than 98% homogeneity and was shown by N-terminal amino acid analysis to be the bona fide product of the 4305-bp SA polC ORF. The physical and catalytic properties of recombinant SA pol III and its responsiveness to inhibitors of the HPUra type were similar to those of Bacillus subtilis (BS) pol III. Comparative structural analysis of the primary structure of SA pol III and the pol IIIs of B. subtilis and the Gram+ relative Mycoplasma pulmonis indicated strong conservation of essential catalytic domains and a novel zinc-finger motif. Comparison of the primary structures of E. coli pol III and these three Gram+ enzymes suggested a specific evolutionary relationship between the pol IIIs of Gram+ and Gram- bacteria.

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Jia Niu / Directed Evolution has emerged as an invaluable tool for advancing protein functions in both research and industry. Our lab has pioneered a directed evolution platform in mammalian cells, utilizing an AAV delivery vector to package a DNA library and linking the biomolecule of interest to AAV production. During my tenure in Prof. Chatterjee's lab, I focused on harnessing our lab’s directed evolution platform, known as Virus-Assisted Directed Evolution of tRNA (VADER), to develop highly efficient tRNAs for genetic code expansion. Additionally, I contributed to the development of the AAV-based selection platform, termed Virus-Assisted Mutational Profiling (VAMP), as a profiling tool. Through the utilization of VAMP, I conducted comprehensive profiling of tRNA and RNA polymerase III promoter sequences. This enabled me to gain insights into regions of flexibility and evolution, ultimately leading to the construction of improved constructs with enhanced activity relative to the starting sequence. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

6-(p-Hydroxyphenylhydrazino) uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific DNA polymerase III (pol III) of Gram+ bacteria such as Bacillus subtilis. Although a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. The project described in this thesis dissertation involves the use of H2-HPUra-like inhibitors to probe the structure and function of the pol III active site. It consists of two separate problems which are summarized below. Production of a potent bona fide dGTP form of inhibitor. A method was devised to successfully convert the H2-HPUra inhibitor prototype to a bona fide purine, using N2-benzyl guanine as the basis. Structure-activity relationships of benzyl guanines carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl) guanine (DCBG) as a compound equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2'-deoxyribonucleoside 5'-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization. The latter experiment identified the site for the binding of the inhibitor's aryl moiety as a distinct site located at a distance of approximately 6-7 Å from the base-paired 2-NH group of a bound dGTP. Attempt to covalently label amino acid residue 1175, a putative participant in inhibitor binding. Azp-12, a point mutation of serine 1175, yields a form of pol III whose inhibitior sensitivity varies specifically as a function of the composition of the para substituent of the inhibitor's aryl ring. On the basis of the latter behavior, residue 1175 was hypothesized to be a residue directly involved in the binding of the inhibitor's aryl moiety. To test this hypothesis, residue 1175 was specifically mutated to either cysteine or lysine, each of which presents a side chain amenable to covalent bond formation with appropriately reactive inhibitor forms. Of the two mutant pol III forms, only the cysteine form (pol III-cys) was catalytically active. The kinetic properties and inhibitor sensitivity profile of pol III-cys identified it as a target suitable for potentially irreversible inhibitor forms containing the following groups in the meta position of the aryl ring: -CH2Br, -CH2C1, and -CH2SH. None of the several inhibitors tested selectively or irreversibly inactivated pol III-cys. Possible bases for the failure of this group of inhibitors and for the redesign of more useful covalently reactive inhibitor forms are considered.