Browsing by Author "Oelgeschlger, Thomas"

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    Biomolecular condensation of intrinsically disordered regions in P. falciparum and H. sapiens transcription factors
    (2022) Karamanof, Leonidas Marthinus; Oelgeschlger, Thomas
    Plasmodium falciparum is still a major cause of disease and death, especially in sub-Saharan Africa. The organism's complex life cycle is tightly coupled to a carefully controlled gene expression programme, and its highly divergent transcriptional machinery suggests unique mechanisms of transcriptional control. Biomolecular condensation, driven by intrinsically disordered regions (IDRs) in proteins, has emerged as a previously underappreciated mechanism of cellular compartmentalisation and regulation. The study of eukaryotic transcription has been revolutionised by the understanding that biomolecular condensates formed by transcription factors play a key role in transcriptional regulation. The study these of so-called transcriptional condensates has so far not addressed the role of the general transcription factors (GTFs) and has not investigated the phenomenon in non-model eukaryotes such as P. falciparum. Here we report on the biochemical purification and functional characterisation of an ensemble of TF-IDR fluorescent protein fusion constructs, using fluorescence microscopy. During the course of this work, we established a standardised data acquisition, analysis and visualisation pipeline for fluorescence microscopy images. Bioinformatic analysis uncovered unique sequence characteristics of the P. falciparum RPB1 C-terminal domain (PfRPB1-CTD) that suggest a functionally divergent role in biomolecular condensation. This work demonstrates that the PfRPB1-CTD, as well as a panel of GTF-IDR fusion proteins, including P. falciparum (Pf) and Homo sapiens (Hs) TATA-binding protein-IDR (TBP), TFIIAαß-IDR and TFIIFß-IDR can drive biomolecular condensation in vitro. Comparative analyses show that PfRPB1-CTD and PfTBP-IDR drive biomolecular condensation at much lower protein concentrations than homologous regions from H. sapiens, suggesting a potential role in organising P. falciparum transcriptional condensates. This work further expands on the characterisation of the IDR from the largest subunit of the Mediator complex, MED1-IDR. Our results underscore its status as a strong driver of biomolecular condensation and further show that the material properties of MED1-IDR condensates are affected by a range of environmental conditions. Finally, we report on the compatibility of P. falciparum and H. sapiens proteins to form mixed condensates. We demonstrate that both PfRPB1-CTD and MED1-IDR exhibit wide-ranging compatibility with GTF-IDR fusion proteins, that are recruited to and concentrated in mixed assemblies. However, while MED1-IDR recruits HsRPB1-CTD to form homogenously mixed transcriptional condensates, PfRPB1-CTD and MED1- IDR form heterogenous mixed condensates, in which the two protein components form separate compartments. This result provides first evidence that transcriptional condensates formed in human cells and in the malaria parasite may have distinct properties and thus may provide a highly promising target for the much needed development of new antimalarial drugs.
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    Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP)
    (2019) Van Der Linden, Lize-Mari; Oelgeschlger, Thomas
    Plasmodium falciparum, the deadliest strain of human malaria, affected 200 million people and resulted in several hundred thousand deaths in 2017 (World Health Organization, 2018). A better understanding of the mechanisms of P. falciparum gene regulation can open novel avenues for the development of much needed new drugs. A key step in eukaryotic gene regulation is the process of transcription, which is largely uncharacterized in Plasmodium. Bioinformatic analysis identified putatuve P. falciparum orthologues of RNA polymerase II general transcription factors (Bing, 2014; Milton, 2017), including a TATA box-binding-like protein, PfTLP. Bioinformatic analysis suggested that PfTLP is a TRF2-type TBP-like protein. However, PfTLP differs in several aspects from previously characterized TRF2-type proteins. These differences are thought to be Plasmodium specific adaptations to the parasite’s intricate life cycle and AT-rich genome. This study investigates two Plasmodium-specific features of PfTLP. Firstly, DNA binding by eukaryotic TATA-box binding protein (TBP) is mediated by four evolutionary conserved phenylalanine residues, two of which intercalate into the DNA. These residues are absent in previously characterized TRF2-type TLPs, and consistent with this, these proteins lack detectable DNA binding activity (Duttke et al., 2014). In contrast, PfTLP, a TRF2-type TLP, has DNA binding activity, and all four of the DNA binding phenylalanine residues are conserved (Bing, 2014; Milton, 2017). The importance of evolutionary conserved intercalating phenylalanine residues F60 and F283 was investigated by generating mutant PfTLP proteins, carrying alanine substitutions, and analysing their DNA-binding properties. The results suggest that while both phenylalanine residues are important for PfTLP DNA-binding, only F60 is critical for stabilization of PfTLP/DNA complexes. Secondly, PfTLP possesses two low-complexity or intrinsically disordered regions (LCR1 and 2), which are absent in TLPs from model eukaryotes. These regions are located at the same positions within the two quasi-symmetrical repeats of the TLP core structure and show a non-random compositional bias towards a limited set of amino acids. A growing body of evidence supports the idea that low complexity or intrinsically disordered proteins mediate liquid-liquid phase separation (LLPS) (Alberti et al., 2019; Brangwynne et al., 2009; Elbaum-Garfinkle et al., 2015; Nott et al., 2015). Bioinformatic analysis revealed that PfTLP LCRs are enriched in asparagine and lysine, and that these regions are well conserved throughout Plasmodium TLPs. PfTLP LCRs were fused to fluorescent proteins and the fusion proteins were functionally characterized in liquid-liquid phase separating assays. The results demonstrate that PfTLP LCR1 is capable of mediating LLPS, at least under certain conditions in vitro.
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    Functional divergence of the RNA polymerase II transcription machinery in Plasmodium falciparum
    (2024) Knopp, Jasmin; Oelgeschlger, Thomas
    This thesis describes the functional characterisation of three Plasmodium falciparum general transcription factors - two TBP family proteins designated the TATA-binding protein (TBP) and TBP like protein (TLP), and the P. falciparum orthologue of general transcription factor IIB (TFIIB). Through the biochemical characterisation of protein-DNA interactions, protein-protein interactions and the biomolecular condensation properties of these transcription factors, this study aimed to provide insight into the molecular mechanisms governing promoter recognition and the regulated assembly of the RNA polymerase II transcription initiation complex in P. falciparum, which are hitherto not understood. The work shows that P. falciparum expresses two highly divergent TBP family proteins that do not possess sequence-specific TATA box-binding activity. Both PfTBP and PfTLP bind DNA with a general preference for A/T-rich sequences, mediated through interactions with the DNA minor groove, without detectable preference for specific sequence motifs. PfTBP and PfTLP are thus unlikely to contribute to the recognition of specific promoter regions within the A/T-rich context of the P. falciparum genome. The study further characterises TBP-independent DNA-binding of PfTFIIB, a Plasmodium-specific feature not seen with TFIIB in well-studied eukaryotes. The data presented here show that PfTFIIB binds to DNA in an unspecific manner. Interestingly, PfTFIIB stimulates the DNA binding activity of PfTBP and PfTLP and forms stable PfTBP-PfTFIIB-DNA and PfTLP-PfTFIIB-DNA nucleoprotein complexes. However, these PfTFIIB interactions do not detectably enhance the sequence-selectivity of PfTBP- or PfTLP-DNA interactions. Thus, recognition of genomic regions at which transcription is preferentially initiated must involve additional factors or may depend to a lesser extent on the recognition of specific core promoter elements by the general transcription machinery. Transcription condensates play an important role in eukaryotic transcription regulation. In this study, the potential role of PfTFIIB in the formation and regulation of P. falciparum transcription condensates is investigated by fluorescence microscopy using a panel of fluorescent protein-tagged PfTFIIB fusion proteins. The work demonstrates that PfTFIIB undergoes condensation at nanomolar concentrations, and partitions with PfTBP and PfTLP into mixed phase separated condensates. Furthermore, assembly and properties of PfTFIIB condensates are shown to be strongly influenced by the presence of DNA and RNA. Interestingly, PfTBP and PfTLP are found to localise in discrete foci in cultured P. falciparum blood-stage parasites. Together, these results provide first evidence for the existence of transcription condensates, mediated by transient multivalent interactions between general transcription factors, in the P. falciparum malaria parasite.
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    Transcriptional regulation in Plasmodium falciparum: characterisation of TBP-like protein
    (2025) Michowicz, Joanna; Oelgeschlger, Thomas
    Malaria persists as a devastating disease resulting in a high burden of morbidity and mortality worldwide. The most severe form of malaria is due to infection with Plasmodium falciparum, accounting for the overwhelming majority of malaria deaths. Despite extensive efforts to curb transmission, recent reports of drug resistance to front-line antimalarial treatments coupled with the continuing lack of effective vaccines implores the production of novel anti-malarials. Both parasite development and pathogenicity are tightly controlled by a highly regulated gene expression program. However, regulation at the level of transcription initiation is hitherto poorly understood. The TATA-binding protein (TBP) is a universal transcription factor required for transcription by RNA polymerase I, II and III. Multiple gene duplication events throughout eukaryotic evolution have resulted in many eukaryotes harbouring more than one TBP family protein. In metazoans, TBP paralogs have been shown to play important roles in cell-specific developmental gene expression programs. However, research into the roles of TBP paralogs in unicellular eukaryotes is extremely limited. Bioinformatic analyses have identified a P. falciparum putative TBP-like protein (PfTLP). Previous research in our laboratory demonstrated that PfTLP has DNA-binding activity and that it harbours two insertions, which are predicted intrinsically disordered regions (IDR1, IDR2), within its structurally conserved TBP DNA-binding domain. In recent years there has been mounting evidence that IDRs mediate liquid-liquid phase separation, a process which has transformed our understanding of cellular organisation and regulation of key cellular processes. The functional relevance of these IDRs is unknown. Therefore, to elucidate their role, this study was divided into three parts: (i) a bioinformatic analysis to investigate the prevalence of disordered regions within Apicomplexan TLPs, (ii) an investigation into the role of these regions in PfTLP DNA binding and (iii) an in-depth characterisation of phase separation by PfTLP IDRs and full-length PfTLP. This study highlights the prevalence of TLPs within Apicomplexa and, moreover, the presence of a conserved IDR2 in diverse Apicomplexan species. Both IDR1 and IDR2 were shown to bind to DNA and analysis of a panel of newly generated PfTLP deletion mutant protein constructs revealed that this DNA-binding activity stabilises PfTLP-DNA complexes. Finally, a systematic characterisation of the phase-separating properties of PfTLP IDRs and full-length PfTLP was carried out, using PfRBP1-CTD and PfTBP-IDR for comparison. It was found that PfTLP-IDR1 readily undergoes liquid-liquid phase separation (LLPS) at low protein concentrations, forming dynamic condensates that homogenously mix with dsDNA. In contrast, PfTLP-IDR2 does not self-assemble in the absence of phase-separating interaction partners. Interestingly, at a physiological protein concentration, full-length PfTLP was found to form clustered assemblies in isolation but formed separate, mixed condensates in the presence of dsDNA, suggesting a role of DNA in the regulation of PfTLP condensate formation. In addition, the interaction of PfTLP with DNA condensates was found to be independent of IDR1 and IDR2, consistent with the DNA-binding activity of the PfTLP structured domain. Thus, this study yields novel insights into the DNA-binding and phase-separating properties of PfTLP that may contribute to the assembly of the RNA polymerase II transcription initiation machinery of this divergent parasite.