Group II introns are large, multi-domain ribozymes that exist in all domains of life. They can catalyze self-splicing reaction that involves excision of introns themselves out of precursor RNAs followed by ligation of flanking exons. Although group II introns are sufficient to carry out the splicing reaction alone, the reaction efficiency could be substantially enhanced by the association of their dedicated protein partners called maturases. Maturases are also reverse transcriptases (RTs) that facilitate the retro-transposition activity of group II introns. Because of their mobility through retro-transposition, group II introns play important roles in eukaryotic evolution. Particularly, group II introns are believed to be the ancestors of eukaryotic spliceosomes, spliceosomal introns, telomerase and non-LTR retrotransposons.
As the first step of understanding the structure, biochemistry and biophysics of RNAs in the context of group II intron system, I started to explore group II intron folding by solving the crystal structure of its folding intermediate (Chapter 2). Group II intron folding is particularly interesting because unlike most other RNAs that are prone to be trapped kinetically in unproductive products along the folding pathway, group II introns fold through a two-step pathway that is free of kinetic traps. The folding intermediate in this two-step pathway is the domain 1 (D1) of group II introns, and after D1 folds, the downstream domains dock rapidly onto the D l scaffold. I solved the crystal structure of a group II intron D1 to 2.95 Å (Chapter 2). This crystal structure explains the strategy of D1 to effectively serve as an on-pathway folding intermediate: the overall scaffold is maintained by a stable five-way junction, and hinge motions at peripheries allow D1 to sample low energy states that could potentially compensate the loss of interactions from downstream domains when D1 is in isolation. This strategy also provides insights on the structural dynamics of RNA molecules in general.
After looking at the structure and structural dynamics of group II intron RNA, as the next step, I started to explore the structure of the protein partner (maturàse) of group II introns (Chapter 3). The maturases have been difficult structural biology targets for over two decades. To tackle this problem, I decided to systematically investigate all the maturases in the group II intron database to search for candidates that are suitable for X-ray crystallography. From the candidates acquired using this method, I was able to solve the first-in-class crystal structure of the RT domain of a group II intron maturase to 1.2 Å (Chapter 3). This crystal structure reveals a scaffold that shares high structural homology to spliceosomal core protein Prp8, therefore provides the first evidence that the protein components from group II introns and eukaryotic spliceosomes are evolutionarily related. The structure also reveals a positively charged surface that is responsible for interacting with group II intron RNA. Additionally, this crystal structure showed a novel loop (α-loop) that might play a role in reverse transcription (Chapter 4). Finally, the maturase RT domain forms a dimer in the crystal structures, and the same interface might mediate the dimerization of the full-length maturase when associated with group II intron RNA.
After solving the crystal structure of the maturase RT domain, I followed up on investigating the potential function of a-loop in reverse transcription reaction (Chapter 4). My hypothesis is that the a-loop is involved in reverse transcriptase processivity because it encloses the active site. RT processivity assay shows that a-loop deletion mutant lost processivity, therefore supporting my hypothesis. Additionally, under the same condition, wide-type maturase has a higher processivity than Superscript IV that is derived from retrovirus M-MLV RT. Therefore, it is likely that the unique a-loop contributes to the superior processivity of group II intron maturases, and potentially in structurally related non-LTR RTs. Besides investigating this α-loop-mediated processivity, I also designed some maturase mutants that uncouple the reverse transcriptase function from the RNA binding function, thus setting up the ground for streamlining the E.r. maturase as a tool enzyme (Chapter 4).
Finally, I also investigated the role of maturases in group II intron splicing and reverse splicing (Chapter 5). I found that maturases not only speed up the splicing reaction, but also switch the splicing pathway from primarily hydrolysis to primarily branching. Specifically, this acceleration of the splicing rate depends on a conserved lysine residue in maturase thumb domain. Additionally, I have found that the reverse-splicing reaction requires the assistance of maturase. The future direction is to understand whether maturase dimerization play a role in maturase-assisted group II intron splicing and reverse-splicing.
|Advisor:||Pyle, Anna Marie|
|School Location:||United States -- Connecticut|
|Source:||DAI-B 79/05(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Biophysics|
|Keywords:||Group II Intron, Maturase, RNA, Reverse Transcriptase, Structural Biology, X-Ray Crystallography|
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