Browsing by Subject "RNA"

Now showing 1 - 8 of 8
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    Assessment and Improvement of Computational Models to Study Biological Catalysis
    (2014-08) Huang, Ming
    A detailed understanding of the molecular mechanisms whereby molecules of RNA can catalyze important reactions such as phosphoryl transfer is fundamental to biology, and of high significance in the development of new biomedical technology. This thesis describes the testing, application and development of quantum models that advance our understanding of the mechanisms of RNA catalysis. Molecular simulations of catalytic mechanisms of RNA require the use of fast, accurate approximate quantum mechanical (QM) models. These models, however, were not necessarily designed and parameterized for biocatalysis. In order to assess the degree to which commonly used approximate QM models are appropriate for biocatalysis applications, a series of models has been tested against a wide range of data sets, including new datasets particularly relevant for RNA catalysis, and compared with high-level benchmark calculations. Results provide new insight into the strengths and weaknesses of these methods, and help to guide next generation model development. We note that both NDDO and SCC-DFTB based QM models fail dramatically in their ability to adequately describe the conformational landscape of DNA and RNA sugar rings. In order to overcome this problem, an empirical sugar pucker energy term has been introduced via multi-dimensional B-spline interpolation of a potential energy surface correction. The corrected semiempirical models closely reproduce the ab initio puckering profiles as well as the barrier of an RNA transesterification model reaction. In addition, a series of RNA transesterification model reactions with various leaving groups have been studied with density-functional calculations in solution to investigate linear free energy relationships (LFERs) and their connection to transition state structure and bonding. These relations can be used to aid in the interpretation of experimental data for non-catalytic and catalytic mechanisms. A driving force in this research has been the development of software infrastructure for scientific computation, including new interfaces to other computational chemistry software, libraries to retrieve information, convert format and apply potentials, and tools for data analysis and visualization.
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    Charge-dependent non-bonded interaction methods for use in quantum mechanical modeling of condensed phase reactions
    (2015-10) Kuechler, Erich
    Molecular modeling and computer simulation techniques can provide detailed insight into biochemical phenomena. This dissertation describes the development, implementation and parameterization of two methods for the accurate modeling of chemical reactions in aqueous environments, with a concerted scientific effort towards the inclusion of charge-dependent non-bonded non-electrostatic interactions into currently used computational frameworks. The first of these models, QXD, modifies interactions in a hybrid quantum mechanical/molecular (QM/MM) mechanical framework to overcome the current limitations of ‘atom typing’ QM atoms; an inaccurate and non-intuitive practice for chemically active species as these static atom types are dictated by the local bonding and electrostatic environment of the atoms they represent, which will change over the course of the simulation. The efficacy QXD model is demonstrated using a specific reaction parameterization (SRP) of the Austin Model 1 (AM1) Hamiltonian by simultaneously capturing the reaction barrier for chloride ion attack on methylchloride in solution and the solvation free energies of a series of compounds including the reagents of the reaction. The second, VRSCOSMO, is an implicit solvation model for use with the DFTB3/3OB Hamiltonian for biochemical reactions; allowing for accurate modeling of ionic compound solvation properties while overcoming the discontinuous nature of conventional PCM models when chemical reaction coordinates. The VRSCOSMO model is shown to accurately model the solvation properties of over 200 chemical compounds while also providing smooth, continuous reaction surfaces for a series of biologically motivated phosphoryl transesterification reactions. Both of these methods incorporate charge-dependent behavior into the non-bonded interactions variationally, allowing the ‘size’ of atoms to change in meaningful ways with respect to changes in local charge state, as to provide an accurate, predictive and transferable models for the interactions between the quantum mechanical system and their solvated surroundings.
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    Early Decay Mechanisms of Brown Rot Wood-Degrading Fungi: Transcriptome Patterns, Cation Dynamics, and Substrate Chemistry Effects
    (2024-05) Anderson, Claire
    Fungi gained the capacity to degrade lignocellulose approximately 295 million years ago when they adapted oxidative enzymes to metabolize lignin – a mechanism still used by white rot fungi. Since then, brown rot fungi have evolved a carbohydrate-selective mechanism that uses reactive oxygen species (ROS) to cause extensive, nonspecific depolymerization in plant cell walls, thus solubilizing the carbohydrates and avoiding lignin removal. Brown rot fungi differentially express genes to create a sequence of decay starting with lignocellulose oxidation via ROS, followed by hemicellulose side chain cleavage and main chain degradation, then cellulose degradation via endoglucanases. It is still unclear how brown rot fungi regulate this elaborate mechanism and avoid subjecting their own enzymes and hyphae to ROS damage. Specifically, the process that turns ROS pathways on at the beginning of brown rot decay has not yet been identified, despite assumptions of an inducible mechanism. Many studies have suggested that the presence of either lignin or hemicellulose may initiate brown rot decay, but this has not been clearly shown experimentally.To address this knowledge gap, I captured the earliest stages of brown rot decay by Rhodonia placenta and analyzed the whole transcriptome at the incipient stage of decay to confirm delayed upregulation of the lignocellulose oxidation genes involved in ROS generation. I also examined the interactions between R. placenta and its lignocellulose substrate in two ways. First, I created a fine-resolution map of the cation translocation dynamics in R. placenta and white rot fungus Pleurotus ostreatus for comparison. Second, to examine the effects of lignin and hemicellulose on brown rot gene expression, I used mutant strains of model plant Arabidopsis thaliana with changes in cell wall chemistry as a substrate for R. placenta and examined the whole transcriptome response of the fungus to these modified lignocellulose substrates. This dissertation contributes more clarity to the transcriptomic details of early brown rot as well as the effects of substrate chemistry on brown rot decay. Understanding brown rot decay mechanisms offers potential to harness these pathways for biotechnology applications as well as to make better predictions about the fate of carbon stored in wood.
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    Gene Expression Within the Fluctuating Life Cycle Stages of Trypanosome Parasites
    (2019-05) Susa, Emily
    Trypanosoma cruzi and Trypanosoma brucei are vector-borne protozoan parasites that cause devastating disease to humans and livestock in South America, North America and Africa. Both parasites have complex life cycles which involve a variety of different environments and nutrient sources within mammalian hosts and arthropod vectors. Transitioning between life cycle stages requires a transformation of morphology, replicative ability, and metabolism, which requires remodeling of mitochondrial and nuclear gene expression. To better understand the complicated genetic factors involved in life stage differentiation in these organisms, we have investigated several aspects of the regulation of gene expression through life stage transitions. In T. brucei, we investigated the role of a putative endoribonuclease, EEP1, on the transition from the mammalian life stage to the insect life stage. Our results indicate that EEP1 does not play a role in the differentiation process, and may serve an entirely unique function. In T. cruzi, we examined the mitochondrial genome which plays a crucial role in metabolism and has been shown to exhibit life-stage specific remodeling in related species. Mitochondrial genome regulation must occur post-transcriptionally in the form of RNA editing, translational control, and stability. Significant changes were detected in mature mRNA abundance between several life stages, and these differences appeared to be correlated with nutrient availability and replication status. Overall, both of these studies provide further understanding of the regulatory processes that govern life cycle transitions in trypanosome parasites.
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    Light-mediated Sexual Dimorphism in Opsin Expression During Spawning in Nematostella vectensis
    (2024-04) Wagner, Starla J.; McCulloch, Kyle J.
    Across animals, opsins are the primary protein responsible for light detection. Currently, there is a large gap in knowledge in the evolutionary history of opsin function and how it correlates with other biological responses like spawning. Cnidarians (jellyfish and anemones) are prime candidates for closing this gap. They are a sister taxon to bilaterally symmetric animals like flies and humans, and so studying their opsin function and expression in non-visual contexts allows for further understanding of how light sensing may have evolved to form modern visual systems. In this experiment, qPCR analysis on the Cnidarian, Nematostella vectensis (the starlet sea anemone), was used to determine the effect of certain wavelengths of light that an animal was exposed to during spawning had on opsin expression levels. The impact of sex and tissue type on these expression levels was an additional area of interest. The data showed that certain wavelengths like blue light were correlated with larger amounts of opsin expression in female mesenteries and tentacles/skin tissue than in male tissue types. This indicates that opsin expression is sexually dimorphic which implies there is a relationship between opsin expression and spawning, something that was previously unknown. Future experiments using RNA-seq will allow for a deeper understanding of this relationship and the proteins involved.
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    Lineage specific reprogramming to blood using a cocktail of transcription factors
    (2010-12) Shaikh, Seema
    A single fertilized cell has the ability to develop into any cell depending on the various cues it responds to. This ability to differentiate into desired cell types can be made use of in the field of developmental biology for studying early embryonic development and for regenerative medicine. Previous work in the lab showed that mouse ES cells engineered with an inducible construct co-expressing the hematopoietic regulatory factors SCL, LMO2 and GATA2 give very efficient hematopoiesis. In monolayer differentiation where hematopoiesis does not occur because the majority of cells differentiate towards ectoderm, expression of these 3 factors diverted cells towards hematopoietic lineage. My work in the lab addressed two questions: Can we optimize the system to obtain hematopoietic progenitors instead of differentiated blood cells? and How does the SCL complex reprogram cells at the molecular level? I added a cocktail of cytokines to the serum free, growth factor free medium to obtain undifferentiated hematopoietic progenitors. A short pulse of induction was sufficient to obtain large number of CD41+, hemoglobin expressing, round semi adherent cells. This treatment gave rise to progenitors of myeloid, erythroid and megakaryocytic lineages proving the multipotent nature of the blood cells that differentiated to both hematopoietic progenitors and committed erythroid cells. To understand the reprogramming potential of the SCL complex at the transcriptional level I performed two RNA sequencing experiments. The first experiment evaluated the early changes and showed that the SCL complex up-regulated many important hematopoietic genes including SCL, LMO2, GATA2, Lyl1 and Gfi1 within 6 hours, but other genes (globins) required a longer period of induction. The second experiment evaluated cells 3 days after a reprogramming pulse. The data showed that the non-reprogrammed (CD41-) cells expressed hematopoietic genes, but at lower levels compared to the fully reprogrammed cells (CD41+) indicating that the cells were not completely reprogrammed. Surprisingly the CD41- cells had higher expression of endodermal genes indicating that the cell could have reverted endoderm, a nearby lineage during the chase period. The data also showed a decrease in the ectodermal genes in both CD41+ and CD41- cells suggesting that the triple construct may be stably erasing the ectodermal program even in non-reprogrammed cells. These results improved the efficiency of the system and shed light on the mechanism of the SCL-LMO2-GATA2 action in lineage specific reprogramming.
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    Reciprocal regulation between the prgQ and prgX operons of pCF10, a conjugative plasmid of Enterococcus faecalis.
    (2011-07) Johnson, Christopher Mark
    Regulation of conjugation of pCF10, a pheromone-response plasmid of Enterococcus faecalis, is a well characterized process that serves as a model for the control of gene expression in bacteria by intercellular signaling. The genes encoded in the pCF10 prgQ operon mediate conjugative transfer of the plasmid in response to a peptide pheromone secreted by plasmid-free E. faecalis cells. The products of the prgX operon, the small RNA Anti-Q and PrgX, the transcriptional repressor of the prgQ promoter, negatively regulate expression of the prgQ operon. Transcription of prgX is initiated at a promoter within the prgQ operon, but oriented in the opposite direction, making transcription of the two operons overlapping and convergent. Each operon encodes a small RNA within its 5' terminus, which is complementary to 5' sequences of the opposing operon. The orientation of the operons and RNA species derived from the 5' terminus of each operon suggested that, in addition to regulation of the prgQ operon by Anti-Q, there may be unrecognized regulatory interactions between the two operons. This led me to undertake a focused study of potential transcriptional and posttranscriptional regulatory interactions between the prgQ and prgX operons. I found that the operons encode mechanisms to negatively regulate each other. Both use a small RNA to reciprocally regulate the expression of downstream genes from the other operon. Notably, each RNA acts via a different mechanism; Qs, derived from the 5' terminus of the prgQ operon, directs posttranscriptional processing of the prgX mRNA by the host factor RNase III. Anti-Q, an RNA derived from the 5' terminus of the prgX operon, negatively regulates transcription elongation of the prgQ operon without the assistance of host-encoded proteins. Additionally, I gained understanding of cis-acting mechanisms that regulate expression of the prgX operon. Specifically, transcription from the prgQ promoter represses the activity of the prgX promoter via a mechanism termed transcriptional interference, and Anti-Q sequences act as an intrinsic terminator, attenuating the expression of prgX mRNA. In addition to elucidating some of the complexity of regulation between the prgQ and prgX operons, this work revealed the high degree of functional efficiency in the RNA sequences of both Anti-Q and Qs. Anti-Q directs its own genesis by acting as an unusual factor-independent termination signal to RNA polymerase and the mature RNA regulates gene expression from the prgQ operon. Qs, for its part, fills at least three roles in the regulation of conjugation, acting as a leader sequence that can attenuate downstream gene expression, a mRNA for the prgQ gene, and a regulatory RNA that directs endonucleolytic processing of its target. Despite the fact that the overlapping sequences between prgQ and prgQ are shared by both operons, the sequences necessary for Anti-Q and Qs to mediate each of their activities are distinct. This raises the possibility that these regulatory functions evolved separately and that the combination of two ancestral regulatory pathways gave rise to the pheromone-response circuit of pCF10. This work has a number of broader implications. It deepens our understanding of how bacteria use RNAs to regulate gene expression. Additionally, the mobile genetic elements of E. faecalis contribute to the evolution of multi-drug resistant E. faecalis and other pathogens. Understanding the molecular mechanisms used by conjugative plasmids may allow the development of novel interventions to prevent this evolution or target bacteria carrying these plasmids.
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    Theoretical studies of RNA catalytic activity.
    (2010-08) Giambasu, George M˘ad˘alin
    RNA possess a diverse ensemble of conformations that interchange on time scales that range from femtoseconds to milliseconds. This conformational variability has a profound effect on RNA function, and has been exploited in RNA molecules such as aptamers, aptazymes, and riboswitches that have been engineered to be allosterically controlled. Central to this work is the questions of how RNA conformational variability affects with its catalytic activity. We employ a set of diverse theoretical and simulation tools, such as molecular dynamics, free energy calculations, and path sampling that greatly extend the capability of structural and molecular biology experiments to reveal atomic level details of RNA energetics and dynamics. Our systems of interest are two prototypical catalytic RNA systems: L1 ligase and Hammerhead ribozymes, both of which are postulated to reach their catalytically active states through complex mechanisms that involve large domains movements, change in base pairing patterns and direct participation of divalent metal ions. In the case of L1 ligase, we reveal the structural basis of its allosteric control that involves an 80 angstrom swing of one of its stems, as well as the role of a flexible active site toward providing catalytic selectivity. In the case of Hammerhead ribozyme, we characterize the conformation and dynamics of several constructs in different ionic environments that lead to catalysis.