Font Size: a A A

Characterization of the seven major polypeptides of the Trypanosoma brucei mitochondrial RNA editing complex

Posted on:2005-01-20Degree:Ph.DType:Dissertation
University:The Johns Hopkins UniversityCandidate:O'Hearn, Sean FrancisFull Text:PDF
GTID:1454390008994625Subject:Biology
Abstract/Summary:
Kinetoplastid RNA editing, post-transcriptional insertion or deletion of uridine residues in mitochondrial transcripts, is catalyzed by a complex of at least seven polypetides. Two of these polypeptides are the adenylylatable RNA ligases designated as bands IV and V. Three of these polypeptides, Bands II, III, and VI contain zinc finger domains as well as a conserved homologous region with the Band VII protein. Band I contains two C-terminal endo/exonuclease domains. I used tryptic peptide sequences generated by Wistar to the proteins and genome databases to clone and express two of the proteins, Bands V and VI. I generated recombinant proteins to all seven of the polypeptides in an attempt to determine specific enzymatic function. With these recombinant proteins, I also produced polyclonal mouse and rabbit antibodies.; RNA interference is a new molecular tool used to silence gene-products by reducing the amount of nascent RNA in the cell in a dsRNA sequence specific manner. I applied the tools of RNAi to 'knock-down' expression of the seven polypeptides and analyzed the results. I found that Bands II, III, IV, VI, and VII are essential for cell growth.; Using the RNAi in conjunction with my antibodies, it was determined that Band III is necessary for the stability of the Band IV ligase. Band III also plays a key structural role in the complex and is necessary for gRNA-directed endonuclease cleavage and U deletion. Band II is necessary for the stability of the Band V ligase and retains the p57 terminal-U-transferase protein. It was further determined that Band II protein is essential for specific enzyme recognition at each step of the U-insertion cycle but is not similarly important for U-deletion.; Additional analysis indicate Band VI is responsible for holding a subset of proteins, Bands I, III, and N together and Band VII is the key protein keeping the entire complex together. Additionally, The insertional ligase, Band V, is redundant and Band IV can ligate both U-deletional and U-insertional editing sites. Results presented demonstrate a specific physical symmetry of the proteins in the complex however the enzymatic functions may overlap.
Keywords/Search Tags:RNA, Complex, Editing, Polypeptides, Band, Proteins, Seven, III
Related items