title: Association of a novel preribosomal complex in Trypanosoma brucei determined by fluorescence resonance energy transfer.
abstract: We have previously reported that the trypanosome-specific proteins P34 and P37 form a unique preribosomal complex with ribosomal protein L5 and 5S rRNA in the nucleoplasm. We hypothesize that this novel trimolecular complex is necessary for stabilizing 5S rRNA in Trypanosoma brucei and is essential for the survival of the parasite. In vitro quantitative analysis of the association between the proteins L5 and P34 is fundamental to our understanding of this novel complex and thus our ability to exploit its unique characteristics. Here we used in vitro fluorescence resonance energy transfer (FRET) to analyze the association between L5 and P34. First, we demonstrated that FRET can be used to confirm the association between L5 and P34. We then determined that the binding constant for L5 and P34 is 0.60 ± 0.03 μM, which is in the range of protein-protein binding constants for RNA binding proteins. In addition, we used FRET to identify the critical regions of L5 and P34 involved in the protein-protein association. We found that the N-terminal APK-rich domain and RNA recognition motif (RRM) of P34 and the L18 domain of L5 are important for the association of the two proteins with each other. These results provide us with the framework for the discovery of ways to disrupt this essential complex.
authors.affiliation: Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, The State University of New York, Buffalo, New York, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Martin
authors.initials: M
authors.lastname: Ciganda
authors.forename: Noreen
authors.initials: N
authors.lastname: Williams
mesh_terms: D020816:Amino Acid Motifs; D031541:Fluorescence Resonance Energy Transfer; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D015800:Protozoan Proteins / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry*; D014346:Trypanosoma brucei brucei
chemical_list: D015800:Protozoan Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C476232:p34 protein, Trypanosoma brucei