Mechanism of RNA/DNA Editing Activity by Single Subunit RNA Polymerase Superfamily Members




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RNA editing in Physarum polycephalum causes insertion of non templated nucleotide(s) at specific positions in the RNA transcript in a co-transcriptional manner. Although Physarum mitochondrial RNA polymerase has been identified as the major player in this whole process and been characterized as well, yet the actual mechanism of RNA editing in Physarum is largely unknown. The unavailability of the information regarding the promoter sequence for Physarum mtRNAP posed further challenge. Since, bacteriophage T7 RNA polymerase as a prototype member of single subunit RNA polymerase shares resemblance in structure and sequence with Physarum mtRNAP, I used T7 RNA polymerase to study the mechanism of RNA editing and further characterization of mtRNAP in vitro. In the process of further characterization of Physarum mtRNAP in terms of its functional domains, I constructed several plasmids containing full length and truncated versions of Physarum mtRNAP gene as well as chimeric product between T7 RNAP and Physarum mtRNAP. These constructs helped us to understand the roles of N-terminus and C-terminus sequences in these polymerases. I also, found the putative promoter sequence for Physarum mtRNAP and confirmed the identity of the promoter sequence by using different constructs containing the putative promoter sequence as well as polymerase sequences of different lengths (with respect to their functional zone). Although there were earlier reports about T7 RNAP adding unwanted extra nucleotide on the DNA and RNA template and efforts were made to prevent this from happening, none of those reports had clear explanation about the mechanism of how and why these activities were observed. I discovered that T7 RNA polymerase indeed have unique editing activity in terms of its ability to add non templated nucleotide at the 3’ end of DNA/RNA, which is similar to the RNA editing observed in Physarum mitochondria. In my research, I characterized the mechanism of this unique editing activity of T7 RNAP (which is different than the regular initiation or elongation activity by the same) and also optimized the parameters responsible for this non templated nucleotide addition. I also observed similar effects/mechanism in other members of single subunit RNA polymerase superfamily, viz., bacteriophage T3 and SP6. Together, all these studies can provide clues towards the evolution of editing mechanism and shed light towards understanding the mechanistic evolution of polymerases from a single origin.



RNA polymerases, RNA editing, Mitochondrial DNA, Cloning, Microbiology


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