Katari, Venkata Subbaraju
Trypanosoma brucei editosomes have a single, bifunctional reaction center - Evidence for a non-collisional reaction mechanism.
Technische Universität, Darmstadt
[Ph.D. Thesis], (2014)
Available under Creative Commons Attribution Non-commercial No Derivatives, 2.5.
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|Item Type:||Ph.D. Thesis|
|Title:||Trypanosoma brucei editosomes have a single, bifunctional reaction center - Evidence for a non-collisional reaction mechanism|
Most mitochondrial transcripts in African trypanosomes are edited to generate translatable transcripts. The reaction is catalyzed by a macromolecular protein complex, the 20S editosome. Editing is characterized by the site-specific insertion and/or deletion of exclusively U nucleotides and in order to catalyze the reaction, editosomes must bind a panel of different substrate pre-mRNAs.
The experiments documented in chapter one verify that 20S editosomes bind different “in vivo-sized” transcripts with nanomolar affinities and association/dissociation rate constants typical for RNA/protein complexes. The editosome/RNA interaction is non-discriminative, thus enabling the interaction with different pre-edited mRNAs as well as with partially edited mRNAs and guide RNAs. Using immunogold-labeling in combination with transmission electron microscopy (TEM) I was able to demonstrate that editosomes have only one RNA substrate-binding site, which suggests that both subtypes of the RNA editing reaction (U-insertion and U-deletion) are catalyzed within a single, bifunctional reaction center.
In chapter two I present the first atomicforce microscopy (AFM)-based pictures of 20S editosomes and 20S editosome/RNA complexes. The data confirm that editosomes have a single RNA binding domain and further demonstrate that editosomes contain a so far unknown “chaperone-type” RNA unwinding activity. Upon RNA binding, transcripts become progressively unwound, ultimately enabling multiple 20S editosomes to interact with one substrate RNA.
RNA editing is a pre-requisite for the survival of Trypanosoma brucei. The life cycle of the parasite involves the cyclic transmission between a mammalian host and the Tsetse fly as the insect vector. Since RNA editing has been shown to be regulated between the two developmental stages, I analyzed in chapter three whether RNA editing is also regulated within the cell cycle of the parasite. Editosome isolates from the G1- and G2-phase of the trypanosome cell cycle were tested for their RNA editing activity. The experiments identified catalytic activity in both phases thus demonstrating that the processing reaction is not cell cycle-regulated.
The basic steps of the editing reaction cycle have been unraveled with the help of an in vitro assay that is per-formed at dilute solvent conditions. However, in vivo the reaction takes place inside the highly “crowded” mitochondrial environment. In chapter four I analyzed the effects of macro-molecular crowding on RNA editing using defined conditions from dilute to semidilute to crowded solvent proper-ties. I was able to demonstrate that the thermodynamic stabilities of the pre-mRNA/gRNA hybrid RNAs differ at these conditions. Crowded solvent properties stabilize the RNA molecules and alter the rate constants for the association and dissociation of the substrate RNAs to editosomes. Ultimately, the processing reaction is inhibited. These results imply that the in vivo reaction cannot rely on a diffusionally-controlled, collision-based mechanism. The data advocate a scenario in which RNA editing is conducted by a “hand-over” or “channeling” of substrate RNAs from one processing machinery to the next.
|Place of Publication:||Darmstadt|
|Classification DDC:||500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie|
|Divisions:||10 Department of Biology > Postranscriptional Gene Regulation and RNA Therapeutics|
|Date Deposited:||14 Jan 2014 16:14|
|Last Modified:||14 Jan 2014 16:14|
|Referees:||Göringer, Prof. Dr. H. Ulrich and Hammann, Prof. Dr. Christian|
|Refereed:||6 December 2013|