Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 104-115). Also available in print.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 96-115).

The accurate prediction of an RNA secondary structure from its sequence will enhance the experimental design and interpretation for the increasing number of scientists that study RNA. While the computer programs that make these predictions have improved, additional improvements are necessary, in particular for larger RNAs. The first major section of this dissertation is concerned with improving the prediction accuracy of RNA secondary structures by generating new energetic parameters and evaluating a new RNA folding model. Statistical potentials for hairpin and internal loops produce significantly higher prediction accuracy when compared with nine other folding programs. While more improvements can be made to the energetic parameters used by secondary structure folding programs, I believe that a new approach is also necessary. I describe a RNA folding model that is predicated on a large body of computational and experimental work. This model includes energetics, contact distance, competition and a folding pathway. Each component of this folding model is evaluated and substantiated for its validity. The statistical potentials were created with comparative analysis. Comparative analysis requires the creation of highly accurate multiple RNA sequence alignments. The second major section of this dissertation is focused on my template-based sequence aligner, CRWAlign. Multiple sequence aligners generally run into problems when the pairwise sequence identity drops too low. By utilizing multiple dimensions of data to establish a profile for each position in a template alignment, CRWAlign is able to align new sequences with high accuracy even for pairs of sequence with low identity. / text

Numerous essential cellular processes, such as gene regulation and tRNA processing, are carried out by structured RNAs. While in vitro most RNAs become kinetically trapped in non-functional misfolded states that render them inactive on a biologically-relevant time scale, RNAs folding in vivo do not share this same outcome. RNAs do indeed misfold in the cell; however, chaperone proteins promote escape from these non-native states and foster folding to functional conformations. DEAD-box proteins are ATP-dependent RNA chaperone proteins that function by disrupting structure, which can facilitate structural conversions. Here, studies with both local and global focuses are used to uncover mechanistic features of DEAD-box proteins CYT-19 and Mss116p. Both of these proteins are general RNA chaperones as they each have the ability to facilitate proper folding of multiple structured RNAs. The first study probes how DEAD-box proteins interact with a simple duplex substrate. Separating the strands of a duplex is an ATP-dependent process and is central to structural disruption by DEAD-box proteins. Here, how ATP is utilized during duplex separation is monitored by comparing ATP hydrolysis rates with strand separation rates. Results indicate that one ATP molecule is sufficient for complete separation of a 6-11 base pair RNA duplex. Under some conditions, ATP binding in the absence of hydrolysis is sufficient for duplex separation. Next, focus is shifted to a more global perspective as the function of Mss116p is probed in the folding of a cognate group II intron substrate, aI5[gamma], under near-physiological conditions. Three catalytically-active constructs of aI5[gamma] are used and catalysis serves as a proxy for folding. Folding of all constructs is promoted by the presence of Mss116p and ATP. In vitro and in vivo results indicate that a local unfolding event is promoted by Mss116p, stimulating formation of the native state. Lastly, orthogonal methods that probe physical features of RNA are used to provide insight into the structural intermediates with which Mss116p acts. / text

Structured RNAs are pervasive in biology, spanning a functional repertoire that includes messengers, regulators of gene expression and catalysts of translation and splicing. From the relatively simple tRNAs and riboswitches to the highly structured ribosomal RNAs, the ability of RNAs to function is dependent on well-defined secondary and tertiary structures. However, studies of RNA folding in vitro have revealed an extreme propensity to form alternative structures, which can be long-lived and interfere with function. In the cell, a diverse array of RNA binding proteins and RNA chaperones guide RNAs towards the correct structure and disrupt misfolded intermediates. Among these proteins, DEAD-box protein family stands out as one of the largest groups, with its members ubiquitously involved in RNA metabolism across all domains of life. DEAD-box proteins can function as both specific and general RNA chaperones by disrupting RNA structures in an ATP-dependent manner. Here I describe my work studying the general RNA chaperone mechanism of the Neurospora crassa protein CYT-19, a model DEAD-box protein and a biological RNA chaperone that is required for efficient folding of self-splicing group I intron RNAs in vivo. After an introduction to DEAD-box proteins and their mechanisms as RNA remodelers (Chapter 1), I will first describe studies of group I intron unfolding by CYT-19, focusing on the effects of RNA tertiary structure stability on CYT-19 activity and targeting to RNA substrates (Chapter 2). I will then describe the characterization of ATP-dependent mechanisms during CYT-19-mediated refolding of the misfolded group I intron (Chapter 3). In Chapter 4, I will present small-angle X-ray scattering (SAXS) studies of structural features of DEAD-box proteins that allow them to efficiently interact with large structured RNA substrates. Finally, I will turn to studies of DEAD-box protein involvement during early steps of RNA compaction and folding, using SAXS and activity-based approaches (Chapter 5). I will conclude with a general discussion of superfamily 2 RNA helicases, which include DEAD-box and related proteins, and their functions and mechanisms as remodelers of structured RNAs and RNPs. / text

When proteins are unable to fold properly in the endoplasmic reticulum (ER), the resultant formation of misfolded proteins causes stress of the ER. Cells with ER stress often have a higher abundance of reactive oxygen species (ROS). Previous studies suggest that ROS could aggravate ER stress by further disrupting the ER protein folding process. More recent studies suggest that the unfolded protein response signaling pathways activated by ER stress could lead to the production of ROS. Such studies lead to the hypothesis that ER stress could be promoted by ROS, and vice versa. The aim of the present study is to test the above hypothesis by studying how ROS could be generated in ER-stressed cells. This is followed by investigating if ROS could increase or decrease the level of ER stress in cells. Finally, the extent of ER stress induced cell death in the presence and absence of ROS is assessed. The treatment of HeLa cells with tunicamycin (Tm), a common ER-stress inducing agent, resulted in the elevation of intracellular ROS that could be detected with the ROS-reactive probe dichlorodihydrofluorescein (DCF), but not dihydroethidium which is relatively specific towards superoxide anion. The Tm-induced elevation of ROS could be prevented by co-incubation of cells with thiol reductants such as dithiothreitol and N-acetylcysteine but not with the free radical scavenger ascorbate. The tunicamycin-induced elevation of ROS level could also be prevented by the over-expression of catalase in HeLa. These data is consistent with the idea that hydrogen peroxide is a major form of ROS produced in Tm-treated cells. In addition to elevation of ROS level, HeLa cells treated with tunicamycin also resulted in the phosphorylation of PERK and eIF2α, and the splicing of XBP-1. In the presence of cycloheximide to inhibit protein synthesis so as to deplete protein substrates for folding in the ER, tunicamycin-induced ER stress was greatly minimized as was evident by the absence of both the phosphorylation of PERK and splicing of XBP-1. However, the phosphorylation of eIF2α and elevation of DCF-detectable ROS remained unaffected. The cycloheximde-resistant phosphorylation of eIF2α could be prevented when cells were co-treated with thiol reductants, or upon the over-expression of catalase. These data suggest that the production of ROS in Tm-treated cells does not require the presence of ER stress as a prerequisite. Furthermore, the ROS so produced could induce phosphorylation of eIF2α without the need to cause ER stress in the first place. The quenching of ROS through the use of thiol reductants, or the over-expression of catalase, had no effect on inhibition of protein synthesis in cells treated with tunicamycin. However, the extent of cell death was significantly increased. The data obtained in this study is not consistent with the idea that ROS is a downstream product of ER stress, capable of inducing more ER-stress by a feedback mechanism. Therefore, a mutually enhancing effect between ER stress and ROS may not exist. The ROS found in stressed cells may serve to extend cellular survival under the condition of continuous stress. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts. The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.