Elucidating the structure and kinetics of the apocytochrome B mRNA/gRNA complex in Trypanosoma brucei mitochondria

Expression of mitochondrial genes in Trypanosoma brucei requires RNA editing of its mRNA transcripts. During editing, uridylates are precisely inserted and deleted as directed by the guide RNA (gRNA) template to create the protein open reading frame. This process involves the bimolecular interaction of the gRNA with its cognate pre-edited mRNA and the assembly of a protein complex. The importance of RNA structure in establishing a functional editing complex is poorly understood. Previous experiments indicate that different mRNA/gRNA pairs can form similar secondary structures suggesting that a common core architecture may be important for editosome recognition and function. Using solution structure probing, we have investigated the structure of the initiating gRNA, gCYb-558, in the mRNA/gRNA complex. The data indicate that the stem-loop formed by the guiding region of the gRNA alone is maintained in its interaction with the pre-edited message. In addition, the data suggest that a gRNA stem-loop structure is maintained through the first few editing events by the use of alternative base pairing with the U-tail. This suggests that the gRNA stem-loop is an important component of the initial editing complex.; In trypansomes, RNA editing of the mitochondrial mRNAs is developmentally regulated in a transcript specific manner. We hypothesize that regulation involves the structure of the mRNA and its ability to interact with its gRNA. Surface plasmon resonance was used to measure the kinetics of gRNA binding for three separate mRNA/gRNA pairs; two mRNA substrates with predicted single stranded anchor binding sites (ABS) and one mRNA substrate with the ABS located within a thermodynamically stable stem-loop. The stability of the mRNA stem-loop appears to affect the gRNA anchor target binding and results in a slower association rate as well as a faster dissociation rate. In contrast, the mRNAs with an open ABS associate with their cognate gRNAs at a much faster rate. In addition, they have a surprisingly slow dissociation rate. This slow dissociation rate may be necessary to allow the editosome protein complexes to recognize and assemble onto the mRNA/gRNA pair.; Editing of the mRNA, results in a progressive lengthening of the anchor helix. Using apocytochrome b (CYb) mRNA and 2 partially edited CYb mRNAs, the kinetic effects of editing for the CYb mRNA/gRNA interaction were investigated. The CYb mRNA forms a stable stem-loop that the gRNA anchor has difficulty invading to base pair at the anchor binding site (ABS). Each editing event appears to result in a decreased dissociation rate that may be important for editing progression. In addition, the U-tail targets one side of the stem-loop for binding and this appears to increase the association rate constant two fold when the gRNA binds the unedited CYb mRNA providing a new and exciting function for the U-tail.