In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates |
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Authors: | Leszek Wawiórka Dawid Krokowski Yuliya Gordiyenko Daniel Krowarsch Carol V Robinson Ishag Adam Nikodem Grankowski Marek Tchórzewski |
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Institution: | 1. Department of Molecular Biology, Maria Curie-Sk?odowska University, Akademicka 19, 20-033 Lublin, Poland;2. Department of Chemistry, University of Oxford, South Parks Rd, Oxford OX1 3QZ, UK;3. Department of Protein Biotechnology, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland;4. Department of Obstetrics & Gynecology, Faculty of Medicine, AlKaser Street, University of Khartoum, Khartoum, Sudan |
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Abstract: | BackgroundThe ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins.MethodsWe have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods.ResultsAll three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)2 form represents the most favorable architecture for parasite P-proteins.ConclusionThe formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1–P2 heterodimeric intermediate.General significanceOn the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role. |
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Keywords: | NAI naturally acquired immunity GAC GTPase Associated Center SEC Size-exclusion chromatography CD circular dichroism BACTH bacterial two-hybrid system MS mass spectrometry MW molecular weight Da daltons |
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