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Characterizing the Role of Ribosomal Protein L7Ae in Archaeal RNase P Catalysis and Exploring the Use of Archaeal RNase P as a Functional Genomics Tool

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2010, Doctor of Philosophy, Ohio State University, Molecular Genetics.

RNase P is an essential ribonucleoprotein (RNP) complex that catalyzes 5’-maturation of tRNAs. It is composed of an RNase P RNA (RPR), the catalytic moiety, and a variable number of RNase P protein (RPP) subunits: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Towards dissecting protein-aided RNA catalysis, our laboratory has successfully reconstituted functional archaeal RNase P holoenzymes by assembling the RPR and four known RPPs. However, since the in vitro reconstituted holoenzymes exhibit lower specific activity (kcat/Km) and optimal temperature for precursor-tRNA (pre-tRNA) cleavage than the native holoenzyme, it is clear that the archaeal RPPs identified so far do not represent the complete suite present in the native version. In this regard, I investigated and demonstrated that ribosomal protein L7Ae, a human RPP38 homolog, is a true component of RNase P from Methanococcus maripaludis (Mma), a mesophilic archaeon. Western analysis of partially purified native Mma RNase P revealed that L7Ae co-elutes with RNase P activity. Addition of recombinant Mma L7Ae to the Mma RPR and four known RPPs in vitro increases the kcat/Km (by 360-fold) and the optimal temperature to that observed with the native holoenzyme. L7Ae is known to bind kink-turns in RNAs. We identified a kink-turn-like motif in the Mma RPR as a binding site of L7Ae by footprinting and mutagenesis studies. The stimulatory effect of Mma L7Ae on RNase P activity is dramatically reduced upon mutating either the conserved nucleotides in this putative kink-turn or conserved amino acids in L7Ae. Thus, L7Ae represents an interesting example of a cellular protein associated with tRNA processing (RNase P), RNA modification (snoRNPs) and translation (ribosomes).

Due to its catalytic RNA moiety and essentiality in all cells, RNase P has been customized for various applications. In addition to pre-tRNA cleavage, bacterial and eukaryal RNase P can also cleave model substrates built from two RNAs. For instance, an RNA termed external guide sequence (EGS) could be designed to form a bipartite pre-tRNA-like molecule that is recognized and cleaved by RNase P. This approach has been used to cleave and inactivate cellular target RNAs by expressing the desired EGSs inside cells. We examined the feasibility of using archaeal RNase P for degrading cellular RNAs and thereby down-regulating their function. I demonstrate that bipartite substrates, comprised of a target RNA and EGS, are efficiently cleaved by in vitro reconstituted archaeal RNase P and that EGSs are recycled, indicating that EGSs can provide multiple rounds of substrate recognition and cleavage. Additionally, our results suggest that higher assay temperatures and the preferential binding of EGS to the substrate (compared to the product) contribute to EGS recycling.

Together, these studies provide new insights into interactions between an RNA-binding protein and a specific RNA structural motif that is critical for archaeal RNase P catalysis. Moreover, we demonstrate EGS- and archaeal RNase P-mediated RNA cleavage in vitro. Results from these studies also furnish critical insights into factors that will influence the success of this RNA-mediated RNA-cleavage strategy as a gene knock-down tool in archaea.

Venkat Gopalan (Advisor)
Mark Foster (Committee Member)
Anita Hopper (Committee Member)
Stephen Osmani (Committee Member)
152 p.

Recommended Citations

Citations

  • Cho, I.-M. (2010). Characterizing the Role of Ribosomal Protein L7Ae in Archaeal RNase P Catalysis and Exploring the Use of Archaeal RNase P as a Functional Genomics Tool [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1290555279

    APA Style (7th edition)

  • Cho, I-Ming. Characterizing the Role of Ribosomal Protein L7Ae in Archaeal RNase P Catalysis and Exploring the Use of Archaeal RNase P as a Functional Genomics Tool. 2010. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1290555279.

    MLA Style (8th edition)

  • Cho, I-Ming. "Characterizing the Role of Ribosomal Protein L7Ae in Archaeal RNase P Catalysis and Exploring the Use of Archaeal RNase P as a Functional Genomics Tool." Doctoral dissertation, Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1290555279

    Chicago Manual of Style (17th edition)