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DNA binding and structural studies of truncated forms of the AreA protein from Aspergillus nidulansReynolds, Lindsey January 1998 (has links)
AreA is a transcription activation protein regulating over 100 genes in Aspergillus nidulans. It is a member of the 4 cysteine zinc finger family, with significant sequence homology in the zinc finger domain with related proteins. As with similar proteins, the zinc finger domain has been identified as the DNA binding motif. Other regions of the protein do not appear to playa role in directly binding DNA. A truncated form of AreA, known as the minimal zinc finger protein, containing the zinc finger domain alone, has been cloned and over-expressed in this study. This domain is sufficient to bind DNA specifically, but weakly. However, the addition of a further 30 amino acids, C-terminal to the minimal zinc finger, containing a highly basic tail is shown to increase the specific binding affinity. Other forms of AreA have been characterised and do not significantly increase the affinity of DNA binding. Identification of a consensus binding sequence by SELEX has also demonstrated that the minimal zinc finger is sufficient to specifically recognise the core sequence GA T A but suggests a degree of tolerance for TAT A. Addition of further regions of the protein do not extend the limits of the recognition sequence or change the consensus sequence. The SELEX experiments did not give any evidence of AreA functioning as a dimer, through proteinprotein interactions. Structural studies of the truncated forms of AreA, by circular dichroism, have suggested the formation of secondary structures for the zinc finger motif similar to those of other proteins and in agreement with the published NMR structure of the AreA zinc finger bound to DNA (Starich et ai., 1998a).
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On the role of ppGpp and DksA mediated control of σ54-dependent transcriptionBernardo, Lisandro January 2006 (has links)
The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.
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Experimental evolution with a global regulator mutant in Escherichia coliSeyll, Ethel 12 September 2014 (has links)
CsrA is a global regulator and the main player of the carbon storage regulator (Csr) network, a well- conserved regulatory network in the bacterial world. CsrA is involved in regulation of many physiological processes, including pathways of central carbon metabolism, biofilm formation, motility and virulence in pathogenic species. CsrA acts at the post-transcriptional level by binding specific sequences on its target mRNAs, leading to mRNA destabilization or stabilization. The majority of studies were analyzing a csrA mutant of E. coli K-12 encoding a truncated form of the CsrA protein, retaining residual activity.<p>This work aims at further characterize the roles of CsrA by deleting the entire csrA gene in a recently isolated strain, the uropathogenic E. coli CFT073 strain. Deletion of csrA leads to a marked growth defect, indicating that this gene, although not essential, is primordial for growth. We performed experimental evolution of csrA deletion mutants. Compensatory mutants totally outcompete the original csrA deletion mutant after six days of culture, indicating that the applied selective pressures are strong. The ÄcsrA and three ÄcsrA evolved mutants were extensively analyzed by combining molecular techniques such as genetics, microscopy and use of fluorescent reporters, and global approaches, including comparative proteomics and whole genome sequencing.<p>Our data indicate that csrA deletion strongly affects central metabolism and energy status, constituting an endogenous metabolic stress that, in turn, induces specific stress responses. This work illustrates the interconnection of multiple regulation networks for responding to specific conditions and demonstrates the flexibility of metabolic network to compensate for genetic perturbations in E. coli.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished
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Breaking the Weak Governance Curse: Global Regulation and Governance Reform in Resource-rich Developing CountriesFerreira, Patricia 11 December 2012 (has links)
There is growing consensus that unless resource-rich developing countries improve their domestic governance systems, rising exploitation of mineral, oil and gas resources may result in long-term adverse developmental outcomes associated with the “resource curse”. Despite the consensus, reforms do not abound. This dissertation investigates the obstacles to such reforms, and the mechanisms and strategies that can possibly overcome these obstacles.
I argue that two trapping mechanisms are binding these countries to a “weak governance curse”. One mechanism is the phenomenon of path dependence, which makes a dysfunctional governance path initiated at a past historical juncture resistant to change over time. The other mechanism is rent-seeking behaviour associated with high resource rents, which creates perverse incentives for political and economic actors to resist reforms.
The Law and Development literature has recently produced a rich body of knowledge on governance reform in developing countries, yet it has largely neglected the potential role of innovative global regulatory mechanisms, beyond development assistance, in this process. I argue that this evolving literature ought to draw from global regulation studies to investigate the interaction between unconventional global regulatory mechanisms and domestic governance reform. In this thesis I analyze whether extraterritorial home country regulations, such as anti-bribery, anti-money laundering and securities disclosure regulations, and transnational public-private partnerships, such as the Extractive Industries Transparency Initiative, may offer institutional opportunities for external and internal actors to facilitate policy reforms in resource-rich and governance-poor countries.
My conclusion is twofold. First, there is reason for cautious optimism regarding the potential for unconventional global regulatory mechanisms to provoke positive feedback effects in domestic governance reform. These mechanisms can open innovative institutional pathways of influence to outsiders and insiders promoting governance reform. Second, instead of searching for a regulatory silver bullet, the most promising way to promote reforms in resilient dysfunctional governance systems is to make use of the wide range of conventional and unconventional mechanisms available. A constellation of regulatory instruments opens up the possibility for outside and inside reformers to benefit from a different policy mix of available mechanisms, depending on the specific circumstances of a given country at a particular time.
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Breaking the Weak Governance Curse: Global Regulation and Governance Reform in Resource-rich Developing CountriesFerreira, Patricia 11 December 2012 (has links)
There is growing consensus that unless resource-rich developing countries improve their domestic governance systems, rising exploitation of mineral, oil and gas resources may result in long-term adverse developmental outcomes associated with the “resource curse”. Despite the consensus, reforms do not abound. This dissertation investigates the obstacles to such reforms, and the mechanisms and strategies that can possibly overcome these obstacles.
I argue that two trapping mechanisms are binding these countries to a “weak governance curse”. One mechanism is the phenomenon of path dependence, which makes a dysfunctional governance path initiated at a past historical juncture resistant to change over time. The other mechanism is rent-seeking behaviour associated with high resource rents, which creates perverse incentives for political and economic actors to resist reforms.
The Law and Development literature has recently produced a rich body of knowledge on governance reform in developing countries, yet it has largely neglected the potential role of innovative global regulatory mechanisms, beyond development assistance, in this process. I argue that this evolving literature ought to draw from global regulation studies to investigate the interaction between unconventional global regulatory mechanisms and domestic governance reform. In this thesis I analyze whether extraterritorial home country regulations, such as anti-bribery, anti-money laundering and securities disclosure regulations, and transnational public-private partnerships, such as the Extractive Industries Transparency Initiative, may offer institutional opportunities for external and internal actors to facilitate policy reforms in resource-rich and governance-poor countries.
My conclusion is twofold. First, there is reason for cautious optimism regarding the potential for unconventional global regulatory mechanisms to provoke positive feedback effects in domestic governance reform. These mechanisms can open innovative institutional pathways of influence to outsiders and insiders promoting governance reform. Second, instead of searching for a regulatory silver bullet, the most promising way to promote reforms in resilient dysfunctional governance systems is to make use of the wide range of conventional and unconventional mechanisms available. A constellation of regulatory instruments opens up the possibility for outside and inside reformers to benefit from a different policy mix of available mechanisms, depending on the specific circumstances of a given country at a particular time.
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Dans les interstices du droit formel : responsabilité sociale des entreprises, soft law et gouvernance contractuelle de la chaine d’approvisionnement mondialeBen Matoug, Khadija 06 1900 (has links)
D’essence interétatique, le droit international public semble fermer ses frontières à toute responsabilité des entreprises transnationales. Cette imperméabilité à la reconnaissance d’une responsabilité juridique per se des pouvoirs économiques globaux n’a cependant pas empêché l’ordre international d’être réactif et d’évoluer en douceur. Face à l’apparition de plusieurs scandales liés à de graves violations des droits de l’homme, notamment dans le cadre des chaînes d’approvisionnement globales, il n’était plus concevable pour l’opinion publique de laisser les entreprises transnationales profiter du vide juridique existant. D’un cafouillis normatif émerge une régulation globale fortement imprégnée par le mouvement de responsabilité sociale des entreprises (RSE). Cette institutionnalisation de la RSE s’appuie sur plusieurs instruments normatifs parmi lesquels les principes directeurs des organisations internationales occupent une place centrale. Ces principes ont vocation à guider les actions des entreprises indépendamment de leur localisation géographique par le biais d’une sorte de toile normative uniforme pour tout le globe. En revanche, la mise en œuvre de cette régulation globale de la RSE n’est possible que par les pressions des acteurs sociaux (société civile et marché) qui tendent à promouvoir la responsabilité des entreprises et facilitent ainsi la transformation de la nature de ses normes. Des normes d’origine privée tendent progressivement à devenir une valeur incontournable dans la gouvernance des chaînes d’approvisionnement globales. En intronisant la RSE dans les relations contractuelles, cette gouvernance signerait l’avènement de nouvelles formulations de la RSE. Cette normativité nouvelle et innovante entend dès lors combler l’espace normatif inoccupé par les États. / Destined, in essence, to regulate interstate relations, public international law seems closed to the idea of recognizing liability of transnational corporations. However, this imperviousness to recognizing legal responsibility per se of global economic powers has not prevented a soft evolution of the international order. Following several scandals related to violations of human rights, some in the context of global supply chains, civil society could no longer allow transnational companies to take advantage of the existing legal vacuum. As a result, a global regulation strongly influenced by the trend of corporate social responsibility (CSR) emerged from a normative muddle. This institutionalization of CSR is based on several instruments, including guiding principles on Business and Human Rights which occupy a central position in this regulatory trend.
It is intended that these principles will apply as a uniform, global normative canvas. Therefore, they should guide actions of companies regardless of their geographic location. The implementation of this CSR global framework is possible due to substantial pressure exerted by non-governmental actors, such as civil society and the market. These actors seek to promote CSR and facilitate the transformation of this regulation. Particularly, private standards are playing an increasingly important role in the governance of global supply chains. By establishing CSR in contractual relations, this governance would mark the advent of new regulatory tools which seek to fill in the public law gaps.
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