Browsing by Author "Oelgeschläger, Thomas"

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    Expression and initial characterisation of the Plasmodium falciparum general transcription factors TFIIB and TLP
    (2015) Bing, Steven; Oelgeschläger, Thomas
    Malaria is a leading cause of morbidity and mortality worldwide, and results in approximately 600,000 deaths annually. The life cycle of the parasite is complex, and has several distinct stages of development. The transitions between these stages are brought about through tightly controlled and highly synchronized changes in gene expression. Plasmodium falciparum causes the most lethal form of malaria in humans. The parasite is particularly virulent as it is able to evade immune detection by the infected host. This virulence is directly related to the expression of variable antigens on the surface of infected red blood cells. The control of gene expression is known to be largely regulated via RNA Polymerase II (RNAPII) transcription initiation, but in P. falciparum the underlying mechanisms have not been determined. This primarily because very little is known about both the key protein factors and DNA elements which guide the assembly of RNAPII components into the transcription initiation complex. Bioinformatics studies have shown that there is very little amino acid sequence conservation between human and Plasmodium RNAPII transcription initiation components. Together with the observation that the Plasmodium genome has an extremely high A+T content, this suggests that Plasmodium may have specific mechanisms to initiate transcription, which could be targeted by novel anti-malarials. The general transcription factor TFIIB and the TBP-like protein (TLP) are key proteins involved in the recognition of the core promoter, and the initiation of RNAPII transcription initiation complex assembly. TFIIB stabilises DNA binding of the primary promoter recognition factor, TATA-box binding protein (TBP), and is involved in promoter recognition through interactions with specific DNA sequences up- and downstream of the TBP DNA binding site. TBP-like protein is a member of the TBP protein family that has been implicated in life cycle stage specific gene transcription initiation in various eukaryotic model organisms. This research study reports the first successful expression and purification of recombinant epitope-tagged Plasmodium falciparum TFIIB and TLP proteins. Preliminary assays demonstrate DNA-binding activity for the recombinant Plasmodium TBP-like protein, and suggest DNA-binding activity in Plasmodium TFIIB protein, which has not been demonstrated before in eukaryotic TFIIB.
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    Regulation of transcription in Plasmodium falciparum, the causative agent of severe malaria: initial characterisation of PfTBP and PfTFIIA
    (2017) Milton, Robert A; Oelgeschläger, Thomas
    Malaria, caused by Plasmodium parasites, remains a leading cause for morbidity and mortality worldwide, resulting in more than 430 000 deaths annually. P. falciparum is responsible for the vast majority of severe malaria cases, accounting for more than 90% of malaria-related fatalities, predominantly in subsaharan Africa. The parasite has a complex life cycle, which involves transitioning between multiple distinct morphologies. The severity of the disease is brought about by the variable expression of parasite proteins on the surface of infected red blood cells. The substantial morphological changes, together with the variable expression of cell surface proteins, are governed by tightly controlled stage-specific changes in gene expression patterns. Understanding the regulatory mechanisms that govern these changes is crucial to fully understanding the parasites biology and pathology at the molecular level, a key step toward identifying targets for the development of much needed novel antimalarial drugs. Ultimately, all gene regulatory mechanisms converge to regulate the assembly and function of the RNA polymerase II (RNAP-II) transcription initiation complex composed of RNAP-II and the general transcription factors (GTFs). Bioinformatics analyses show that the RNAP-II GTFs in P. falciparum have greatly diverged from those studied in other eukaryotes, suggesting the existence of parasite-specific gene regulatory mechanisms, which have so far not been studied. This research project concerns the structure and function of P. falciparum TBP, TLP and TFIIA, key proteins involved in core promoter recognition, the first step in RNAP-II transcription initiation complex assembly. The work provides strong evidence for the existence of two different PfTFIIA complexes containing different PfTFIIA-γ subunits. The data further demonstrate that PfTBP and PfTLP DNA-binding activities differ distinctly from the classical TBP-DNA interactions seen in other eukaryotes and demonstrate interaction with and stimulation of PfTBP and PfTLP DNA-binding activity by one of the two PfTFIIA complexes. The work represents a first step towards understanding the regulation of transcription initiation in P. falciparum, gives first insights into Plasmodium-specific features, and provides a solid foundation for further investigations into this crucial aspect of malaria biology.
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    Transcription regulation in Plasmodium falciparum : functional characterisation of general transcription factor IIB
    (2016) Talvik, Gertrud; Oelgeschläger, Thomas
    Plasmodium falciparum is the causative agent of the most severe form of malaria and continues to pose challenges to international healthcare, with high mortality rates and emergence of drug-resistant strains. Plasmodium falciparum has multiple sexual and asexual lifecycle stages within its Anopheline mosquito and human hosts, accompanied by distinct morphological changes. The complex lifecycle, along with the ability to adjust rapidly to different environmental niches, is governed by highly regulated and tightly synchronised changes in stage-specific gene expression. In eukaryotes, regulation of RNA Polymerase II transcription initiation is one of the main mechanism of gene expression control. Past research has revealed the presence of crucial elements of RNA Polymerase II (RNAPII) transcription machinery in P. falciparum, however, the precise transcription initiation mechanisms in P. falciparum remain to date undescribed. Bioinformatics studies have found very little homology between human and P. falciparum transcription factors. Furthermore, because of the extreme bias toward A/T in the Plasmodium genome, TATA-box and other core promoter elements that direct transcription initiation, cannot be determined with confidence in bioinformatics studies. Functional characterisation of the key protein factors involved in transcription initiation is a first important step towards the identification of core promoter elements and could reveal currently unknown eukaryotic transcription initiation mechanisms or new anti-malarial targets. In eukaryotes, TATA-binding protein (TBP) and transcription factor IIB (TFIIB) are the key protein factors involved in the core promoter recognition and RNAPII preinitiation complex (PIC) assembly. TBP nucleates PIC assembly by binding the TATA box, thereafter the TBP-TATA complex is further stabilised by TFIIB. In addition, TFIIB has a crucial role in RNAPII recruitment and transcription start site selection and is therefore deemed indispensable in eukaryotic transcription. P. falciparum TBP is the only PIC component in P. falciparum that has been functionally characterised to date, albeit to a very limited extent. This research study reports the successful expression and purification, as well as initial characterisation of DNA-binding activity of P. falciparum TBP and TFIIB. We observe PfTBP binding at multiple locations on putative P. falciparum promoters. We further report PfTBP-independent binding activity of PfTFIIB, that has not been previously observed under the conditions and has implications for novel DNA-binding mechanisms of PfTFIIB. Furthermore, we conclusively demonstrate the formation of a PfTBP-PfTFIIB-promoter ternary complex.