We have shown that two trypanosome-specific RNA binding proteins, P34 and P37, play an essential role in the ribosomal biogenesis pathway in T. brucei. This represents a novel finding since the ribosomal pathway is highly conserved. To exploit this observation for the ultimate goal of targeting the T. brucei ribosomal pathway in chemotherapy, we must understand the mechanism by which these proteins act in the pathway. In recent work, we have shown that P34 and P37 interact with both L5 and 5S rRNA, the highly conserved components of the preribosomal particle. Loss of P34/P37 leads to a decrease in 5S rRNA levels and other effects in ribosomal formation. The objective of this proposal is to determine the critical residues involved in this interaction and develop a method to identify molecules that disrupt this interaction. Our hypothesis is that the molecular interactions of these proteins will provide a valid target for intervention in ribosomal assembly in T. brucei.
The specific aims of the project are to: 1. Determine the critical molecular determinants on L5 and P34/P37 that are required for participation in the T. brucei preribosomal complex. 2. Determine the critical regions on 5S rRNA that interact with L5 and P34/P37 in the T. brucei preribosomal complex. 3. Determine whether the interaction of P34/P37 with L5 and 5S rRNA is required for trafficking of the complex to the nucleolus. 4. Develop a FRET (fluorescence energy transfer) based screen for the disruption of the interaction between P34/P37, L5, and 5S rRNA in the preribosomal particle. Differences in ribosomal structure and function between bacteria and the infected human host have provided targets for macrolide and ketolide antibiotics. Since this preribosomal particle is unique to kinetoplastids and play an essential role in the ribosomal structure and biogenesis in these organisms, mechanistic studies with this complex will allow us to define a strategic target for chemical intervention.
The protozoan parasites, the Kinetoplastids, include the organisms Trypanosoma brucei, Trypanosoma cruzi and Leishmania, which cause serious diseases in much of the world (African sleeping sickness, Chagas'disease and leishmaniasis, respectively). This study will examine a unique preribosomal complex occurring in these parasites with the goal of developing an assay to identify drugs that target the parasite but not host complex.
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