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Structural Biology of RNA by X-ray Crystallography, Chemical Probing, and Cryo-EM
Full text linkThere are a wide variety of non-coding RNAs that fold into well-defined 3D shapes and play important roles in the cell. Despite the importance of these non-coding RNAs in biology, the field of RNA structural biology is not as well-developed as protein structural biology. The main goal of this dissertation is to elucidate the structures of RNA molecules using several approaches. Other work on nucleic acid-related proteins is also presented.The first part of the dissertation focuses on the group II intron RNA. A crystal structure of the group II intron in the intermediate lariat-3′ exon state was determined to elucidate the mechanism of the second step of splicing and led to a model of the second step in which several junction nucleotides undergo dynamic rearrangements. These dynamic rearrangements are supported by splicing assays of mutants and SHAPE chemical probing. The SHAPE data also revealed that κ-κ′, a tertiary interaction in a different part of the intron, has dynamics that are necessary for splicing.Chapter 4 looks at the mechanism of selective fidelity in diversity-generating retroelements, a class of genetic elements that can generate a large amount of sequence variability in a protein. This work shows that selective fidelity was due to the low catalytic efficiency of the reverse transcriptase and depended on certain substituents in the nucleobase template.The next part of the dissertation explores the use of bacterial nanocompartments as a chaperone for cryo-electron microscopy (cryo-EM) structure determination of RNA . First, a high-resolution cryo-EM structure of a thermostable bacterial nanocompartment is presented, illustrating several of its interesting features. Second, a method to assemble RNA inside a nanocompartment is demonstrated and a cryo-EM dataset of this complex was collected, resulting in a 5 Å reconstruction of the encapsulated RNA.Chapter 7 explores the structure and mechanism of a DNA phosphorothioation complex. This complex is active in vivo and the recombinantly purified proteins bind to DNA in vitro. A cryo-EM dataset of this complex was collected and resulted in a 5 Å density map
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Fluorescent Labeling of RNA and DNA on the Hoogsteen Edge using Sulfinate Chemistry
Full text linkWe have devised a single pot, low-cost method to add azide groups to unmodified nucleic acids without the need for enzymes or chemically modified nucleoside triphosphates. This involves reacting an azide-containing sulfinate salt with thenucleic acid, leading to replacement of C–H bonds on the nucleobase aromatic rings with C–R, where R is the azide-containing linker derived from the original sulfinate salt. With the addition of azide functional groups, the modified nucleicacid can easily be reacted with any alkyne-labeled compound of interest, including fluorescent dyes as shown in this work. This methodology enables the fluorescent labeling of a wide variety of nucleic acids, including natively folded RNAs, under mild conditions with minimal effects upon biochemical function and ribozyme catalysis. To demonstrate this, we show that a pair of labeled complementary ssDNA oligonucleotides (oligos) can hybridize to form dsDNA, even when labeled with multiple fluorophores per oligo. In addition, we also demonstrate that two different group II introns can splice when prelabeled internally with fluorophores, using our method. Broadly, this demonstrates that sulfinate modification of RNA is compatible with ribozyme function and Watson–Crick pairing, while preserving the labile backbone
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Investigation of Tetrahymena Group I Intron Structural Dynamics
Full text linkGroup I introns are a class of catalytic RNAs that can perform a self-splicing reaction. This thesis will examine the large-scale conformational changes of three Tetrahymena group I intron (TET) states. A combination of mutational analysis and cryogenic electron microscopy (cryo-EM) was used to determine the dynamics associated with the L9/P5 mutation (∆L9) in which a long-range tertiary contact that affects the structure-function of TET is knocked out. This mutation allowed for the visualization of dynamics that were not seen in the previously published models of the wild-type (wt) construct. There are three different states of TET modeled: an intermediate form in which the intron is connected to the 5` exon, but the 3` exon is spliced; a second step (S2) state where the intron is base pairing to the ligated exons, and finally, a post-second step (post-S2) state in which the intron is fully spliced. Understanding the structural changes during the splicing of ∆L9 TET provides specific insights into the structure-function relationships of group I introns and general mechanisms of RNA catalysis and dynamics. The structural biology methods developed to facilitate these experiments have also expanded the use of cryo-EM as a general tool for studying many different RNAs. Furthermore, by increasing our knowledge of RNA catalysis, we gain a more comprehensive grasp of these essential biological macromolecules and their role in the origins of life on Earth
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Diverse Chemical Modification of Nucleic Acids with Sulfinate Salts and Structural Studies of Plant Viroids
Full text linkMy co-authors and I have devised a simple, low-cost method to modify RNA with sulfinate salts that can directly add almost any desired functional group under mild conditions. Existing methods of RNA modification have relatively limited applicability due to constraints on the size of the RNA and the lack of diversity of possible modifications. This chemistry modifies the Hoogsteen edge of nucleobases and is done as a single pot reaction. It can be applied to RNA or DNA of any size, as well as to individual nucleotides. Sulfinate salts can modify RNA with a broad range of functional groups, such as fluorophores, biotin and medicinally relevant small molecules such as trifluoromethyl groups. This methodology enables the exploration of diverse chemical groups on RNA that can potentially confer protection from nucleases, allow for efficient delivery of nucleic acids into cells, and act as new tools for the investigation of nucleic acid structure and function. Prior to working on the chemical modification of RNA, I attempted to solve the structure of a viroid using cryo-electron microscopy (cryo-EM) and x-ray crystallography. Viroids are infectious RNAs that target plants, including important food crops such as potatoes, apples and avocados. They are 240 to 400 nucleotides long, single stranded, covalently closed circular RNAs. Viroids, incredibly, do not encode protein and have no DNA replication intermediate. Of the secondary structures that have been experimentally determined, two are predicted to fold into higher order tertiary structures: the peach latent mosaic viroid (PLMVd) and the chrysanthemum chlorotic mottle viroid (CChMVd). Biochemical evidence suggests that these tertiary interactions are essential for infectivity. Many attempts were made to solve a viroid structure with both x-ray crystallography and cryo-EM, but I was ultimately unsuccessful due to the viroid’s persistent aggregation
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Structural Insights Into RNA Splicing
Full text linkThe formation of branched lariat RNA is an evolutionarily conserved feature of RNA splicing reactions for both group II and spliceosomal introns. In both splicing systems, the lariat is formed in the first of two transesterification reactions as a result of the 2' hydroxyl of a conserved bulged adenosine within the intron attacking the phosphate backbone at the 5' splice site. In the second step, the 3' hydroxyl of the 5' exon attacks the 3' splice site, resulting in ligation of the exons and release of the intron as a lariat. Due to the mechanistic and structural similarities, it is believed that group II introns and the spliceosome share a common ancestor. Though many structures have been published of both group II introns and the spliceosome, the precise structural requirements for splicing in both systems remain unknown. In the case of group II introns, all of the structures published prior to the work in this dissertation are of a primitive group IIC intron from the bacterium Oceanobacillus iheyensis (O.i.). This intron splices through a hydrolytic pathway and therefore does not provide a comprehensive understanding of branch formation, which severely limits the conclusions that can be applied to our understanding of the spliceosome. This dissertation describes various experiments that have been performed to better understand how group II introns and the RNA components of the spliceosome active site are able to properly fold and place substrates within the single active site for the two subsequent reactions. Two new crystal structures of the lariat-forming IIB intron from the brown algae Pylaiella litoralis, P.li.LSUI2, at different stages of splicing are outlined in this dissertation: the intermediate lariat–3' exon and post-catalytic lariat. Two different arrangements of the catalytic triplex, the residues which create the active site scaffold, are observed in these structures. However, examination of all published O.i. crystal structures reveals the same catalytic triplex arrangement throughout splicing. Therefore, the rearrangement that occurs in the more evolved P.li.LSUI2 intron is likely facilitating the transition between the stages of splicing and relevant to branch formation and exon ligation in the spliceosome. These structures are also the first in which domain VI is visualized. Domain VI is integral to the branching pathway as it contains the first-step nucleophilic residue. For the first time, domain II can be seen forming two tetraloop-receptor interactions, -' and -', with domain VI proximal to the bulged adenosine. These tertiary interactions appear to stabilize domain VI. However, disrupting either or both interactions simultaneously results in a stalling of the second step. Additionally, iridium hexamine binds in the major groove of domain VI and promotes exon ligation. Taken together, this suggests that domain VI is dynamic and plays an important role in facilitating both steps of splicing
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Structural Insights into RNA Splicing in Group II Introns
Full text linkGroup II introns are a class of ribozymes capable of self-excision from a nascent pre-mRNA sequence via two sequential transesterification reactions. Due to the mechanistic and structural similarities, it is believed that the group II intron and the spliceosome share a common evolutionary ancestor. Splicing is a critical post-transcriptional modification that can be both beneficial, through alternative splicing, and detrimental, in the case of errors in the splicing process, to organisms. The work entailed in this dissertation aims to elucidate the structural elements that are required for accurate and productive RNA splicing. In the first part of this dissertation, the structure of a bacterial pre-catalytic intron was determined in order to understand the architecture necessary to catalyze the first step of splicing. The crystal structure of this intron prior to the first step revealed the intact 5′ splice sit that exhibited a sharp, kinked phosphate backbone, which positions the scissile phosphate alone into the active site and promote the fidelity of the splicing reaction. The second part of this dissertation focused on the crystal structure of a eukaryotic group II intron to determine what structural elements are required to activate the intron for lariat formation. Surprisingly, DII was shown to have an integral role in interacting with DVI, with the identification of a novel tertiary interaction, π-π′ linking the helix of DVI adjacent to the bulged adenosine to a loop of DII. The third section details the crystal structure of the pre-2s group II intron. This structure reveals conserved junction nucleotide rearrangements within the catalytic triplex compared to previous group II intron structures. This suggests these junction nucleotides undergo rearrangements in order to facilitate the transition to the next step of splicing. Finally, the fourth project entails the biochemical and structural characterization of a novel GUAAY pentaloop identified in the lariat forming group II intron crystal structure. Based on the findings of this project, the pentaloop represents a new class of RNA tertiary interaction
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Structural biology of group II intron splicing and retrotransposition
Full text linkGroup II introns are DNA sequences that are interspersed throughout the genomes of organisms from all three domains of life. In many cases these intron genes interrupt coding sequences referred to as exons. In order for the exons to be expressed properly, the intervening intron sequence must first be removed. Once transcribed into pre-mRNA, the scattered introns become catalytic ribozymes and are able to efficiently excise themselves and ligate the flanking exons. These RNA molecules contain an active site capable of binding the catalytic metal ions required to perform two sequential transesterification reactions that cut out the intron sequence and paste the exons together. This mechanism is identical to the one used by the spliceosome to process spliceosomal introns from pre-mRNA in eukaryotes. The mechanistic similarities combined with conserved structural elements supports the hypothesis that group II introns and the spliceosome share a common evolutionary ancestor. In addition to their splicing function, group II introns also act as selfish mobile genetic elements known as retrotransposons. These active retroelements contain an open reading frame for a protein called the maturase. When expressed. this protein acts as a folding chaperone by binding specifically to the intron RNA to promote splicing activity. The maturase is a multifunctional protein containing reverse transcriptase and endonuclease domains allowing the group II intron/maturase complex to invade dsDNA through a target primed reverse transcription mechanism. Sequence homology and mechanistic similarities have evolutionarily linked group II introns to non-LTR retroelements, which make up approximately 34% of the human genome. Some of these retroelements are still active and are the cause of many genetic diseases. The focus of this dissertation is the structural study of group II introns to elucidate the mechanism of splicing and retrotransposition. Using cryo-EM, I have been able to obtain density maps at 5.8 Å resolution for a group II intron while splicing and 4.8 Å resolution for a group II intron actively invading dsDNA. The 4.8 Å map represents the first of its kind for any retroelement
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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