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Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP)
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...
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| Language: | English |
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Department of Molecular and Cell Biology
2020
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| _version_ | 1867613218000601088 |
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| access_status_str | Open Access |
| author | Van Der Linden, Lize-Mari |
| author2 | Oelgeschlger, Thomas |
| author_browse | Oelgeschlger, Thomas Van Der Linden, Lize-Mari |
| author_facet | Oelgeschlger, Thomas Van Der Linden, Lize-Mari |
| author_sort | Van Der Linden, Lize-Mari |
| collection | Thesis |
| description | 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. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/31322 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:32:38.580Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/31322 Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP) Van Der Linden, Lize-Mari Oelgeschlger, Thomas Molecular and Cell Biology 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. 2020-02-25T11:42:39Z 2020-02-25T11:42:39Z 2019 2020-02-25T08:24:53Z Master Thesis Masters MSc http://hdl.handle.net/11427/31322 eng application/pdf Department of Molecular and Cell Biology Faculty of Science |
| spellingShingle | Molecular and Cell Biology Van Der Linden, Lize-Mari Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP) |
| thesis_degree_str | Master's |
| title | Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP) |
| title_full | Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP) |
| title_fullStr | Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP) |
| title_full_unstemmed | Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP) |
| title_short | Functional Characterization of Plasmodium falciparum TATA-box binding-like Protein (PfTLP) |
| title_sort | functional characterization of plasmodium falciparum tata box binding like protein pftlp |
| topic | Molecular and Cell Biology |
| url | http://hdl.handle.net/11427/31322 |
| work_keys_str_mv | AT vanderlindenlizemari functionalcharacterizationofplasmodiumfalciparumtataboxbindinglikeproteinpftlp |