Browsing by Subject "Catalytic RNA"
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Item Characterization of folding and misfolding of the Tetrahymena thermophila group I ribozyme(2013-05) Mitchell, David III; Russell, Rick, 1969-The functions of many cellular RNAs require that they fold into specific three-dimensional native structures, which typically involves arranging secondary structure elements and stabilizing the folded structure with tertiary contacts. However, RNA folding is inherently complex, as most RNAs fold along pathways containing multiple intermediates, including some misfolded intermediates that can accumulate and persist. Our understanding of the origins and structures of misfolded forms and the resolution of misfolding remains limited. Here, we investigate folding of the Tetrahymena intron, an extensively studied RNA folding model system since its initial discovery decades ago. The ribozyme variant predominantly misfolds, and slow refolding to the native state requires extensive structural disruption. Paradoxically, the misfolded conformation contains extensive native structure and lacks incorrect secondary and tertiary contacts despite requiring displacement of a native helix, termed P3, with incorrect secondary structure to misfold. We propose a model for a new origin of RNA misfolding to resolve this paradox, wherein misfolded ribozyme contains within its core incorrect arrangement of two single-stranded segments, i.e. altered topology. This model predicts a requirement for P3 disruption to exchange the misfolded and native topologies. We mutated P3 to modulate its stability and used the ribozyme's catalytic activity to show that P3 is disrupted during the refolding transition. Furthermore, we demonstrate that unfolding of the peripheral tertiary contacts precedes disruption of P3 to allow the necessary structural transitions. We then explored the influence of topology on the pathways leading to the misfolded and native states. Our results suggest that P3 exists in an earlier pathway intermediate that resembles the misfolded conformation, and that P3 unfolds to allow a small yet significant fraction of ribozyme to avoid misfolding. Despite being on a path to misfolding, the decision to misfold depends upon the probability of disrupting P3 and exchanging topology at this intermediate. Additionally, we show that having a stable P3 in the unfolded ribozyme allows almost complete avoidance of misfolding. Together, these studies lead to a physical model for folding and misfolding of a large RNA that is unprecedented in its scope and detail.Item DNA target site recognition by the Ll.LtrB group II intron RNP(2011-08) Whitt, Jacob Tinsley; Lambowitz, Alan; Browning, Karen S.; Iverson, Brent L.; Paull, Tanya T.; Yin, WhitneyMobile group II introns are retroelements that site-specifically insert into DNA target sequences. The group II intron mobility pathway is mediated by a ribonucleoprotein particle (RNP) composed of excised intron RNA and an intron-encoded protein (IEP). The intron lariat inserts at a specific DNA target sequence and is then reverse transcribed by the IEP. Both the intron RNA and IEP are required for DNA target site recognition. I have identified the contact sites within the IEP responsible for recognition of two key positions in the DNA target, T+5 and T-23. IEP recognition of T+5 in the 3'-exon is required for endonuclease cleavage of the bottom-strand of the DNA target site, which generates a primer used for initiation of reverse transcription of the intron. The T+5 base is contacted by G498 in the LtrA DNA-binding domain and nearby residues, particularly K499, potentially bolster this interaction. Recognition of T-23 in the distal 5'-exon is required for initial recognition of the DNA target site by the RNP. The T533 side-chain contacts the T-23 base and the L534 side-chain may also contribute to recognition through hydrophobic interactions with the C5 methyl group. A mutant, L534H, that switches target site specificity to T-23G has been characterized. In order for the RNP to make these and other contacts in the 5'- and 3'-exons simultaneously, the DNA must be bent. I have dissected the role of DNA bending in the intron mobility pathway and found that the DNA is bent at two progressively larger angles as the reaction proceeds. The predominant bend angle at earlier time points places the bottom-strand DNA cleavage site at the protein endonuclease active site. The predominant bend angle of later time points places the cleaved DNA site at the RT domain active site for initiation of reverse transcription of intron cDNA. Finally, in a practical application of group II intron mobility, I have used reprogrammed group II introns ("targetrons") to target two genes in Bacillus subtilis to demonstrate the suitability of targetron technology for gene targeting in the Gram-positive Bacillus genus.Item Engineered regulation of an RNA ligase ribozyme(2001-08) Robertson, Michael Paul; Ellington, Andrew D.Item Group II intron and gene targeting reactions in Drosophila melanogaster(2011-08) White, Travis Brandon; Lambowitz, Alan; Bull, James J.; Macdonald, Paul M.; Paull, Tanya T.; Stevens, Scott W.Mobile group II introns are retroelements that insert site-specifically into double-stranded DNA sites by a process called retrohoming. Retrohoming activity rests in a ribonucleoprotein (RNP) complex that contains an intron-encoded protein (IEP) and the excised intron RNA. The intron RNA uses its ribozyme activity to reverse splice into the top strand of the DNA target site, while the IEP cleaves the bottom DNA strand and reverse transcribes the inserted intron. My dissertation focuses on the Lactococcus lactis Ll.LtrB group II intron and its IEP, denoted LtrA. First, I investigated the ability of microinjected Ll.LtrB RNPs to retrohome into plasmid target sites in Drosophila melanogaster precellular blastoderm stage embryos. I found that injection of extra Mg2+ into the embryo was crucial for efficient retrohoming. Next, I compared retrohoming of linear and lariat forms of the intron RNP. Unlike lariat RNPs, retrohoming products of linear intron RNPs displayed heterogeneity at the 5’-intron insertion junction, including 5’-exon resection, intron truncation, and/or repair at regions of microhomology. To investigate whether these junctions result from cDNA ligation by non-homologous end-joining (NHEJ), I analyzed retrohoming of linear and lariat intron RNPs in D. melanogaster embryos with null mutations in the NHEJ genes lig4 and ku70, as well as the DNA repair polymerase polQ. I found that null mutations in each gene decreased retrohoming of linear compared to lariat intron RNPs. To determine whether novel activities of the LtrA protein contributed to the linear intron retrohoming 5’ junctions, I assayed the polymerase, non-templated nucleotide addition and template-switching activities of LtrA on oligonucleotide substrates mimicking the 5’-intron insertion junction in vitro. Although LtrA efficiently template switched to 5’-exon DNA substrates, the junctions produced differed from those observed in vivo, indicating that template switching is not a significant alternative to NHEJ in vivo. Finally, I designed and constructed retargeted Ll.LtrB RNPs to site-specifically insert into endogenous chromosomal DNA sites in D. melanogaster. I obtained intron integration efficiencies into chromosomal targets up to 0.4% in embryos and 0.021% in adult flies. These studies expand the utility of group II intron RNPs as gene targeting tools in model eukaryotic organisms.Item Mechanistic studies of CYT-19 and related DExD/H-box proteins on folding of the Tetrahymena group I ribozyme(2008-05) Bhaskaran, Hari Prakash; Russell, Rick, 1969-DExD/H-box proteins are a diverse class of proteins that are implicated in RNA and RNP remodeling. They have sequence homology to DNA helicases and share conserved ATPase domains, suggesting that they use the energy of ATP binding and hydrolysis to mediate conformational rearrangements in RNAs. In the past, the action of DExD/H-box proteins has been characterized primarily on simple model substrates such as small RNA duplexes. It is not known how DExD/H-box proteins manipulate structured RNA, what determines target specificity and what molecular events follow their action. Here, using the well-characterized Tetrahymena group I intron ribozyme, I performed kinetic and thermodynamic studies to understand the mechanism of CYT-19 and related DExD/Hbox proteins. CYT-19 has been shown previously to facilitate the folding of several group I and group II introns. I demonstrated that CYT-19 acts as a chaperone, accelerating the re-folding of a long-lived misfolded species of the Tetrahymena group I ribozyme to its native state. Further characterization of this reaction gave insights into how CYT-19 achieves this action; CYT-19 partially unfolds the misfolded ribozyme and allows it to fold again along the same pathway that exists in the absence of CYT-19. In addition to acting on the misfolded state, CYT-19 also acts on the native state, but this action is largely obscured under stabilizing conditions for the native state because the action is inefficient under such conditions. However, under conditions where the native state is destabilized, the native ribozyme was indeed shown to be partially unfolded by CYT-19. By acting on either species, CYT-19 sets up a steady state of unfolding, and the distribution is shifted from equilibrium to kinetic control, increasing the relative populations of conformations that are kinetically preferred during folding. The efficiency of action seems to correlate with the stability of the ribozyme. These activities are not restricted to CYT-19; the DExD/H-box proteins Mss116p and Ded1 were demonstrated to possess similar activities. Together, these studies give important insights into the mechanisms of action for this ubiquitous class of proteins and have implications for all structured RNAs in cells.