Master of Science, The Ohio State University, 2016, Microbiology

Riboswitches are cis-acting regulatory elements that respond to a variety of signals. The S box (SAM-I) riboswitches are S-adenosylmethionine (SAM)-responsive RNA regulatory elements located at the 5' untranslated regions of genes involved in the biosynthesis and transport of methionine and SAM. Binding of SAM to the riboswitch RNA results in repression of downstream gene expression, primarily via transcriptional attenuation. The Bacillus subtilis metK gene, which encodes SAM synthetase, is an atypical member of the S box regulon. Unlike the other 10 S box regulated transcriptional units in B. subtilis, which are induced under methionine starvation conditions that result in low SAM levels, metK is expressed only when methionine concentrations are high and SAM concentrations are low. The metK leader region contains additional conserved sequences 5' and 3' of the S box riboswitch, designated the Upstream (US) and Downstream (DS) boxes, respectively. These sequences are highly conserved in Bacillales sp. The US and DS box elements were shown previously to base-pair in the absence of SAM. Extensive site-directed mutagenesis revealed a core region within the US and DS box elements that are sensitive to alterations in both sequence and base-pairing potential. Additionally, mutations in these sequences result in reduced transcript stability when methionine concentrations are high and SAM concentrations are low in vivo. We hypothesize that metK is regulated by two separate mechanisms. The S box riboswitch regulates expression by sensing the concentration of SAM to either allow transcription to occur when SAM pools are low or cause transcription termination when SAM pools are high. Regulation at the level of transcript stability is proposed to be mediated by an unknown trans-acting factor that binds to the base-paired US and DS boxes when methionine concentrations are high and SAM concentrations are low. In this study, two complementary approaches were employed to ident (open full item for complete abstract)

Committee: Tina Henkin Ph.D. (Advisor); Irina Artsimovitch Ph.D. (Committee Member); Jane Jackman Ph.D. (Committee Member); Ruiz Natividad Ph.D. (Committee Member) Subjects: Microbiology

Bachelor of Science (BS), Ohio University, 2024, Chemistry

In a world where antibiotic resistance is on the rise, the pursuit of novel bacterial drug targets is increasingly important. The T-box riboswitch mechanism, which is involved in regulating the ability for Gram-positive bacteria to make proteins, is a promising target for a novel antibiotic. This study attempts to further understand this mechanism of transcription antitermination and termination in terms of the cofactors that modulate its outcome. The effects of two polyamine molecules, spermine and spermidine, on the riboswitch were analyzed using in-vitro transcription (IVT) assays and denaturing electrophoresis gels. The majority of this work focuses on optimizing the procedure of analysis, and this procedure was finally used to compare the transcription outcomes of the IVT assays that used the two different polyamines. The study concludes that spermine and spermidine differentially modulate the T-box riboswitch transcription outcomes, and that a hypothesized complex formation could have physiological significance in the bacterial cell. Further research is required to elucidate the reasoning behind this difference, but making slight changes to the procedure will likely help make this distinction.

Committee: Jennifer Hines (Advisor); Lauren McMills (Advisor) Subjects: Biochemistry; Chemistry

Bachelor of Science (BS), Ohio University, 2024, Chemistry

Due to the ever-growing health concern of antibiotic resistance, there is a need for novel drug development that can target bacteria in a way that is not resisted. In Gram positive bacteria, one of these potential targets is the T-box Riboswitch, which is a regulatory, non-coding region of RNA involved in amino acid regulation. Within the T-box Riboswitch is an antiterminator region, which dictates whether or not the downstream genes are transcribed. The genes that are regulated by the T-box riboswitch are directly involved in protein synthesis. The antiterminator is stabilized by the binding of uncharged tRNA, and when unbound forms the more stable terminator form, in which transcription is terminated. Drug design can be employed to target the antiterminator to prevent antitermination from occurring and cause the bacterial cell to die due to lack of protein synthesis. This thesis explores fragment-based computational docking studies to determine compounds that bind to the antiterminator with specificity and in regions that could potentially result in an inhibitory effect on antitermination. A compound library of 180 amino acid R groups were prepared and docked to the antiterminator as well as a control model without the tRNA-binding bulge region. Of the initial 180 amino acid R groups, 47 were determined to bind to the antiterminator in regions that may lead to inhibition with more specificity than in the control model. Additionally, 10 peptide links that were produced from these 47 compounds were further docked and each showed some level of binding to the regions of interest in the antiterminator. The results of this project indicated 47 amino acids that could potentially be used as building blocks for drug synthesis, in addition to 10 peptides that may produce an inhibitory effect on the antiterminator. Further studies can be performed on these compounds, such as fluorescence assays and transcriptional assays, to further te (open full item for complete abstract)

Committee: Jennifer V. Hines (Advisor) Subjects: Biochemistry; Chemistry

Doctor of Philosophy (PhD), Ohio University, 2022, Chemistry and Biochemistry (Arts and Sciences)

The T-box riboswitch is an attractive RNA drug target found in gram-positive bacteria. The work described herein details the design, synthesis, and bioactivity of novel conformationally restricted small molecules that were developed in an effort to target the antiterminator structure of the T-box riboswitch. Compounds were designed by use of in silico docking studies. Select compounds were then synthesized as racemates and their bioactivity against the T-box antiterminator was tested, which allowed for the identification of potential lead compounds that were synthesized as pure enantiomers. A fluorescence monitored in vitro transcription assay performed on these enantiopure triazole analogs revealed enantioselective binding interactions with the T-box riboswitch antiterminator, as well as statistically significant inhibition of tRNA-induced transcriptional readthrough.

Committee: Stephen Bergmeier (Advisor); Jennifer Hines (Advisor); Ronan Carroll (Committee Member); Shiyong Wu (Committee Member); Mark McMills (Committee Member) Subjects: Biochemistry; Biology; Chemistry; Organic Chemistry

Master of Science in Biomedical Engineering (MSBME), Wright State University, 2022, Biomedical Engineering

21% of U.S adults experienced mental illnesses in 2020. Nearly 1 in 4 active- duty military personnel showed signs of mental health conditions in 2014 [89]. Mental health can be identified in the body by different biomarkers. These biomarkers potentially could be controlled by riboswitches, which could help mental illnesses and regulate diseases. Riboswitches are desirable in these cases due to responding without affecting vital functions. Riboswitches are located in mRNA and switch “ON” or “OFF” depending on the concentration of a biomarker [13]. In this research, riboswitches were re-engineered to take a known riboswitch and control its response in the presence of a biomarker. This was done by computationally changing PreQ1, a known riboswitch that has the smallest aptamer, and then experimentally testing against biomarkers, dehydroepiandrosterone-sulfate (DHEA-S), Serotonin, Cortisol, Dopamine, Epinephrine, and Norepinephrine. A total of 7 variant riboswitches were tested in this research, 4 created computationally and 3 created in research [53]. The results from these variants showed that variants 1 and 2 had different responses to DHEA-S then the expected PreQ1 response. A dose response test confirmed this by having a downward trend as DHEA-S concentration increased. In conclusion of this research, riboswitches can be re-engineered to have a different response to biomarkers but keep the same structure

Committee: Tarun Goswami D.Sc. (Advisor); Ulas Sunar Ph.D. (Committee Member); Jaime E. Ramirez-Vick Ph.D. (Committee Member) Subjects: Biomedical Engineering

Master of Science (MS), Ohio University, 2022, Chemistry and Biochemistry (Arts and Sciences)

The T-Box Riboswitch is an antibiotic target affecting Gram positive bacteria. The riboswitch senses and regulates many pathways regarding protein synthesis. Wanting to build peptides to inhibit the T-box riboswitch, we first created a library of 260,002 peptides. The peptides were docked to a conserved sequence in the T-box riboswitch and the top 11 peptides were chosen. Chosen peptides were chemically synthesized using the Fmoc/ T-Butyl synthesis and were characterized by MALDI TOF/TOF and RP-HPLC. Initial in-vitro studies show that the peptides will bind to the RNA, though they have not shown to be selective.

Committee: Jennifer Hines (Advisor); Justin Holub (Committee Member); Benjamin Bythell (Committee Member) Subjects: Biochemistry

Doctor of Philosophy (PhD), Ohio University, 2022, Chemistry and Biochemistry (Arts and Sciences)

Antibiotic resistance is a global threat beside the ongoing pandemic by SARS-CoV-2. The number of deaths due to antibiotic-resistant infections is increasing at an alarming rate. The COVID-19 pandemic has already claimed millions of deaths worldwide. Fighting against antibiotic-resistant superbugs and the SARS-CoV-2 has become a challenge. A significant amount of research is going on to develop the vaccine and small molecule antiviral and antibacterial therapeutics targeting proteins. Fortunately, novel non-coding regulatory RNA targets have been identified for developing new antibacterial and antiviral drugs such as bacterial T-box riboswitch, RNA thermometers, and viral stem-loop II motif. T-box riboswitch can control the transcription or translation of amino acid-related genes in bacteria by forming unique interactions between tRNA and mRNA. RNA thermometers (RNATs) are temperature-responsive riboswitches that control the translation based on temperature sensing thus controlling the interaction with the mRNA and 16S rRNA. In Shigella dysenteriae, three RNATs, i.e., ompA, shuT, and shuA, have been discovered. ompA RNAT controls the translation of outer membrane protein A. shuT, and shuA RNAT controls the translation of two proteins that are crucial to the bacterial heme utilization system. The Stem-loop II motif (S2M) is a highly conserved RNA element found in most coronaviruses, astroviruses, and picornaviruses that plays a potential role in viral replication and invasion. The RNA structure plays a significant role in its regulatory function for all of these potential therapeutic targets. Consequently, it is essential to examine the factors that affect the RNA structure and RNA-RNA interaction. Despite having limited building blocks, RNA has diverse functions in the cells. Base protonation and protonated base pairs often occur in RNA when interacting with other biomolecules, thus could play a critical role in vital biological processes. Diff (open full item for complete abstract)

Committee: Jennifer Hines (Advisor) Subjects: Biochemistry; Biology; Genetics

Bachelor of Science (BS), Ohio University, 2021, Chemistry

As the threat of antibiotic resistant infections and outbreaks looms, there has been a reinvigorated interest in identifying new therapeutics to target alternative targets in species primed for developing resistance. One such target is the antiterminator sequence of the T-box riboswitch, an important regulatory motif that acts as an `on' switch for important protein synthesis genes in Gram-positive bacteria. The antiterminator is kinetically favored in transcription of T-box regulated genes, but is thermodynamically unfavored to its counterpart terminator sequence, which contains many of the same nucleotides and prevents gene expression through transcription termination or enveloping the Shine-Dalgarno sequence, preventing translation. The antiterminator is stabilized through interaction with the acceptor end of uncharged tRNA, and as such is responsive to the cellular concentration ratio of charged and uncharged tRNA. As a thermodynamically unstable and highly conserved regulatory element, the T-box antiterminator has been the focus of drug-design efforts to create ligands that would preclude or destabilize tRNA binding to the antiterminator, disrupting protein biogenesis ultimately leading to cell death. In an effort to devise a new primary, high-moderate throughput compound screening to find small molecules which bind to the antiterminator mechanism of the T-box riboswitch with high specificity, this thesis investigates a hybrid assay combining computational and experimental techniques. Computational docking of libraries of compounds using a receptor grid developed from the antiterminator NMR solution structure (PDB: 1N53) is used to identify a selection of compounds with favorable chemical features which bind to the antiterminator with high selectivity and strong bonding values. These compounds can then be tested in a single temperature fluorescence assay against three similar, but structurally disparate models based on the T-box antiterminator to identify li (open full item for complete abstract)

Committee: Jennifer V. Hines Dr. (Advisor) Subjects: Biochemistry; Chemistry

Doctor of Philosophy (PhD), Ohio University, 2021, Chemistry and Biochemistry (Arts and Sciences)

The T-box riboswitch is a cis-acting regulator RNA element found primarily in the 5′ untranslated region of Gram-positive bacterial mRNAs that code for aminoacyl synthetases and other amino acid-related genes. Many of these genes are essential to the survival of the respective bacteria. The T-box riboswitch functions as a two-part system containing an aptamer domain and expression platform that together regulate transcription or translation. A gene-specific uncharged tRNA functions as the effector ligand by binding in the aptamer domain resulting in transcription readthrough (or translation initiation). The presence of the T-box riboswitch only in bacterial and not mammalian cells makes it an interesting drug target, however targeting specific RNA constructs remains a major challenge. Identifying small molecules that can bind discriminately to specific RNA secondary and tertiary structures requires strategically designed prediction models. In this study, combinatory compound screening using molecular modeling and moderate throughput assays was used to identify `hit' compounds that could be studied further with the potential to design ideal small molecule inhibitors. The potential role of T-box riboswitch agonists like polyamines, which are ubiquitous in binding to nucleic acids, to mimic in vivo conditions in vitro was investigated to help identify compounds of interest early in the screening process. Using optimized primary screening assays, a natural products library, RNA-targeted synthetic compounds, and computational methods (e.g., docking, molecular dynamics); a strategy for developing a pharmacophore model for the T-box riboswitch was established. The combinatory screening approach developed could be used for other structured RNA targets of interest. A pharmacophore workflow was designed with iterative improvements assisted by molecular dynamic simulations to obtain a validated pharmacophore model. The molecular dynamic simulations were initially optimized usi (open full item for complete abstract)

Committee: Jennifer Hines PhD (Advisor) Subjects: Biochemistry; Chemistry

Master of Science, The Ohio State University, 2020, Biochemistry

DNA origami is a collection of DNA strands assembled into nanometer-sized devices which have potential uses in several pharmaceutical and biotechnological applications. A major limitation in the current iterations of DNA origami machines is that the constructs have a highly specific response to nucleic acids, thus limiting the range of ligands the nanodevices can sense and respond towards. Riboswitches are a class of RNA elements that regulate mRNA through binding with a diverse range of ligands. Once a ligand is bound to the RNA, it induces a conformational change that sequesters or exposes a sequence known as the regulatory platform. Through engineering the regulatory platform to be complementary to DNA origami binding sequences, a system was devised in which a riboswitch acts as an adaptor molecule that enables the DNA origami to sense small ligands. In this study, DNA hinges were repurposed to anneal to the regulatory platform of SMK, a small SAM binding riboswitch. Through monitoring SAM binding to SMK with 3H-SAM size exclusion assays, we planned to validate the premise of this novel riboswitch-DNA origami system. SAM binding assays gave conflicting results, causing the current state of the project to be inconclusive. However, there are several optimizations that may be pursued in future studies.

Committee: Jackman Jane Ph.D (Advisor); Henkin Tina Ph.D (Advisor) Subjects: Biochemistry; Microbiology; Molecular Biology; Nanotechnology

Doctor of Philosophy, The Ohio State University, 2020, Microbiology

Riboswitches are cis-acting regulatory RNAs that are found in the 5ʹ untranslated region (UTR) of the genes they regulate. These elements modulate gene expression by structural rearrangements in response to a wide variety of physiological signals. The S-adenosylmethionine (SAM)-responsive S-box RNAs are one of the most prevalent classes of riboswitches. S-box riboswitches are found primarily in Firmicutes and regulate the expression of genes involved in methionine and SAM metabolism. These riboswitches consist of an aptamer domain that binds SAM and an expression platform that undergoes a SAM-dependent conformational rearrangement to regulate the expression of downstream coding regions. When the intracellular level of SAM is high, SAM binds to the aptamer domain and stabilizes a terminator helix to cause premature termination of transcription. When the SAM level is low, a competing antiterminator helix forms, which allows transcription of the downstream genes. Bioinformatic analyses have suggested that a rarer class of S-box riboswitches may regulate gene expression at the level of translation initiation in some non-Firmicutes. Indeed, in this study, we identified a translational S-box element in the metI gene of Desulfurispirillum indicum. We demonstrate that this RNA undergoes SAM-dependent changes in the structure of the expression platform and the efficiency of ribosome binding, which are consistent with the regulation at the level of translation initiation. We hypothesized that the translational and transcriptional S-box riboswitches that are located in the same gene in different organisms would regulate expression using different regulatory strategies. Regulatory mechanisms of riboswitches have been shown to be influenced by the kinetics of ligand interaction. A major goal of this work was to compare the ligand binding properties of the transcriptional and translational metI S-box riboswitches. We show that despite the structural similarity, the transcri (open full item for complete abstract)

Committee: Tina Henkin (Advisor); Michael Ibba (Committee Member); Irina Artsimovitch (Committee Member); Venkat Gopalan (Committee Member) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2019, Biochemistry Program, Ohio State

T box riboswitches regulate the expression of amino acid-related genes by monitoring the aminoacylation status of specific tRNAs. Most T box RNAs share a common structural organization that includes three helical domains (Stems I, II, and III), a pseudoknot element (Stem IIA/B), and mutually exclusive terminator and antiterminator helices (or sequestrator and antisequestrator helices for translational regulation). Despite the generally shared architecture, each T box riboswitch responds to a specific cognate tRNA the amino acid identity of which usually corresponds with the function of the regulated gene. Direct base pairing between the T box RNA and the tRNA anticodon and acceptor end act as the primary specificity determinants for cognate tRNA recognition. However, it has been demonstrated that these interactions are not sufficient for cognate tRNA selection. We hypothesize that other interactions between the riboswitch RNA and tRNA, unique features of the tRNA, and amino acid-specific structural variation in the T box RNA contribute to cognate tRNA selection. While most T box RNAs contain all of the structural elements, unique classes of T box riboswitches lack some of the conserved features. In the Ultrashort (US) Stem I class of ileS T box riboswitches, the Stem I helix lacks the apical region that is conserved in canonical T box RNAs, such that the Specifier Sequence is located in the terminal loop, but all of the other conserved structural features are maintained. The primary goal of this research was to determine the role of the conserved structural elements in the US T box RNA for specific tRNAIle binding. Of particular interest was the function of Stem II and Stem IIA/B, as these elements are highly conserved in T box RNAs and are important for riboswitch function in vivo, but their role in the regulatory mechanism remained unknown. In the current study, we demonstrated that both Stem II and Stem IIA/B of the US RNA contribute to tRNAIle affinity. Ad (open full item for complete abstract)

Committee: Tina Henkin (Advisor); Mark Foster (Committee Member); Jane Jackman (Committee Member); Karin Musier-Forsyth (Committee Member) Subjects: Biochemistry; Microbiology

Doctor of Philosophy, The Ohio State University, 2018, Molecular, Cellular and Developmental Biology

Many aminoacyl-tRNA synthetase genes and other amino acid-related genes in Firmicutes are regulated at the level of premature transcription termination by the T box riboswitch. When the nascent transcript (leader RNA) of these genes interacts with a specific uncharged tRNA, an antiterminator element is stabilized which prevents formation of a mutually exclusive transcription terminator helix upstream of the coding sequence. Specificity of the leader RNA-tRNA interaction is determined primarily by a three nucleotide Specifier Sequence that is complementary to the anticodon of the cognate tRNA. An additional base pairing interaction occurs between the acceptor end of uncharged tRNA and a bulge in the antiterminator helix. Most T box leader RNAs share a common structural organization that includes several helical elements (Stems I, II, and III) and a pseudoknot (Stem IIA/B). The Bacillus subtilis glyQS gene, which encodes glycyl-tRNA synthetase, is a natural deletion variant that lacks Stem II and the pseudoknot. The terminal region of Stem I contains two highly conserved sequence motifs that interact for form a platform that stacks with the tRNA D- and T-loops, also known as the tRNA elbow. The sequence motifs within the Stem I platform exhibit a pattern of covariation that varies based on amino acid class, and glycyl genes such as glyQS comprise a separate class. Here, the roles of the glyQS Stem I platform and other Stem I structural elements in tRNA binding and antitermination are investigated. A glyQS variant that contains a Stem I platform that matches the consensus for the T box gene family is characterized and further investigated for its role in tRNA specificity. The glycyl Stem I platform discriminates against non-cognate tRNAs that contain a large variable loop, while the consensus Stem I platform does not. Additional competition experiments with non-cognate tRNAs reveals that there are likely to be additional mechanisms of tRNA specificity that a (open full item for complete abstract)

Committee: Tina Henkin PhD (Advisor); Charles Daniels PhD (Committee Member); Mark Foster PhD (Committee Member); Anita Hopper PhD (Committee Member) Subjects: Microbiology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2016, Microbiology

Bacterial cells regulate gene expression in response to a variety of signals. Regulation can occur at each level of gene expression including, but not limited to, transcription initiation, transcription attenuation and translation initiation. T box riboswitches are found in 5' untranslated leader regions upstream of amino acid related genes in Gram-positive bacteria. T box leader RNAs form several conserved structural elements including three stem-loops (Stem I, II and III), a Stem IIA/B pseudoknot, and mutually exclusive terminator and antiterminator helicies. T box riboswitches regulate gene expression at the level of transcription attenuation by interacting directly with a specific tRNA; uncharged tRNA promotes antitermination. This mechanism was identified through genetic analysis of the Bacillus subtilis tyrS gene, which encodes tyrosyl-tRNA synthetase and is expressed during normal growth. TyrS mischarges tRNATyr with D-tyrosine (D-Tyr), which inhibits growth and biofilm formation due to misincorporation of D-Tyr into proteins. B. subtilis produces D-Tyr and lacks known mechanisms to prevent mischarging. The B. subtilis genome has a second gene that encodes tyrosyl-tRNA synthetase, tyrZ, which is expressed when tyrS is mutated. The tyrZ gene is preceded by the dtrR gene, which encodes a MarR-like transcriptional regulator, and a T box riboswitch. We sought to determine how tyrZ gene expression is regulated and the role of TyrZ. We showed that DtrR represses transcription initiation of the dtrR-tyrZ operon by binding to an operator sequence within the promoter. The T box riboswitch also regulates tyrZ expression. We demonstrated that TyrZ is more selective against D-Tyr compared to TyrS, and that tyrZ expression permits growth and biofilm formation in the presence of D-Tyr. We concluded that tyrZ, which we showed is expressed during biofilm formation, may protect cells against D-Tyr during growth and biofilm formation. During analysis of tyrS (open full item for complete abstract)

Committee: Tina Henkin (Advisor); Irina Artsimovitch (Committee Member); Natividad Ruiz (Committee Member); Mark Foster (Committee Member) Subjects: Microbiology

Doctor of Philosophy (PhD), Ohio University, 2016, Chemistry and Biochemistry (Arts and Sciences)

The T box riboswitch is a regulation mechanism at the level of transcription or translation which controls expression of amino acids related genes, including a lot of essential genes, in many bacteria. The T box riboswitch interacts with cognate tRNAs and senses their aminoacylation status. A charged cognate tRNA allows formation of the thermodynamically more stable terminator structure which induces transcription termination. An uncharged tRNA stabilizes the alternative antiterminator structure and prevents formation of the terminator. Transcription proceeds and leads to expression of the downstream gene(s). The T box riboswitch is a novel and promising drug target since multiple genes essential to bacterial survival are regulated by this mechanism in many pathogenic bacteria. In order to further study the T box riboswitch mechanism and screen a synthetic ligands library, a fluorescently monitored multi-round in vitro antitermination assay with an enhanced throughput was successfully developed and comprehensively evaluated. Using this assay, the effects of molecular crowding, spermidine and DMSO on the T box riboswitch function were studied and 304 ligands were screened. A total of nine ligands showed specific inhibition to the tRNA-induce antitermination. Combining melting temperature analysis and structural probing, the binding of spermidine to the antiterminator was also characterized.

Committee: Jennifer Hines PhD (Advisor) Subjects: Biochemistry; Chemistry; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2015, Molecular, Cellular and Developmental Biology

Riboswitches are cis-acting RNA regulatory elements located in the 5' untranslated region of a gene. These elements modulate gene expression by structural rearrangements in response to an array of physiological signals. T box riboswitches regulate expression of amino acid-related genes by responding to the aminoacylation status of a specific tRNA that matches the amino acid identity of the regulated gene. Most T box RNAs function at the level of transcription attenuation. A terminator that prevents transcription of the downstream gene forms when aminoacylation of the cognate tRNA is high, whereas uncharged tRNA promotes stabilization of an antiterminator that prevents termination and therefore increases gene expression. T box riboswitches are typically composed of three conserved helical domains, designated Stem I, II, III, the Stem IIA/B pseudoknot, and the competing terminator and antiterminator elements; these domains include conserved primary sequence and several secondary structure elements. However, the predicted structure of many T box riboswitches from the phylum Actinobacteria differ from those found in other phyla. A major goal of this research was to characterize the unusual T box RNAs found in Actinobacteria. These riboswitches are present in ileS genes, and were divided into three groups based on the arrangement of the Stem I domain: canonical Stem I; Ultrashort Stem I (US); and Unusually Structured Stem I Region (USSR). The US and USSR domains lack conserved elements in the canonical Stem I that were previously thought to be essential for T box riboswitch regulation. In addition, most of T box riboswitches in Actinobacteria are predicted to regulate gene expression at the level of translation initiation instead of transcription attenuation. In the current study, we demonstrated that several T box RNAs from Actinobacteria are functional in vitro and undergo structural rearrangements and changes in ribosomal binding in response to uncharge (open full item for complete abstract)

Committee: Tina Henkin (Advisor); Irina Artsimovitch (Committee Member); Venkat Gopalan (Committee Member); Anita Hopper (Committee Member) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2012, Microbiology

A variety of mechanisms that regulate gene expression have been uncovered in bacteria. Riboswitches are cis-acting regulatory sequences that reside typically in the untranslated regions of bacterial mRNAs. Riboswitches serve as genetic regulatory switches that sense and respond specifically to environmental signals to regulate expression of the downstream gene, typically in the absence of any protein factor. The S box riboswitch is a transcription termination control system found mostly in Gram-positive bacteria that regulates the expression of many genes involved in sulfur metabolism. The S box genes are characterized by the presence of a set of highly conserved primary sequence and secondary structural elements in the untranslated leader region upstream of the regulated coding sequence. SAM, the molecular effector of the S box riboswitch, is synthesized from methionine and ATP. Expression of the majority of the S box genes is induced during methionine starvation (when SAM pools are low) and is repressed in the presence of methionine (when SAM pools are high). In spite of high sequence and structural conservation, a few S box leader RNAs from Bacillus subtilis fail to exhibit typical S box gene regulation and variation is seen in response to SAM both in vivo and in vitro. This work examines the leader RNA elements that contribute to the observed S box variability, with a special focus on the metK leader RNA. Investigation of the metK leader RNA was performed using biochemical and genetic techniques. We modulated in vivo SAM pools without removing methionine from the growth medium and provided evidence for a SAM-dependent change in metK gene expression in vivo. Phylogenetic analyses revealed the presence of unique sequence elements, the Upstream (US) and Downstream (DS) boxes, that are highly conserved in the metK leader RNAs in several Firmicutes. Using RNase H assays, we showed that these regions are involved in a base-pairing interaction that is stabilized in th (open full item for complete abstract)

Committee: Tina Henkin M (Advisor) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2012, Biochemistry Program, Ohio State

Regulation of gene expression is an essential process that organisms employ for growth and survival. The activation or repression of genes is critical as it allows the cell to monitor environmental signals and adapt to changes in nutrient availability or environmental conditions. One method of regulation involves RNA elements termed riboswitches. Riboswitches are conserved RNA elements that regulate gene expression by modulation of the RNA structure. Typically, a structural change in the RNA occurs in response to environmental signals such as temperature, small RNAs, or small molecules. Most of the known riboswitches modulate gene expression in response to a small molecule effector. Recognition of the effector causes a structural rearrangement that prevents or promotes the formation of a regulatory structure such as an intrinsic transcriptional terminator. Riboswitches that regulate gene expression at the level of translation undergo a structural rearrangement that can occlude or expose the ribosomal binding site (RBS). In this work, we focused our analysis on the L box riboswitch that regulates gene expression during transcription in Bacillus subtilis and is predicted to regulate at the translational level in Escherichia coli. This conserved RNA regulates the expression of lysine biosynthetic genes in response to cellular lysine concentrations. Previous work revealed that the lysC leader RNA from B. subtilis promotes premature transcription termination in the presence of lysine and can discriminate against lysine analogs. Analysis of the leader region in vivo indicated that lysC expression is repressed when the RNA is transcribed in the presence of high lysine. Here we investigate the conserved sequence and structural features of the lysC leader riboswitch to elucidate the features that are required for lysine binding and the required structural transition. We have identified variants of the lysC leader RNA that stabilize the termination conformation in the absence (open full item for complete abstract)

Committee: Tina Henkin PhD (Advisor); Juan Alfonzo PhD (Committee Member); Don Dean PhD (Committee Member); Mark Foster PhD (Committee Member) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2009, Ohio State Biochemistry Program

This work reports the study of relationships between structure and function in two RNA-based elements of life: archaeal RNase P and the bacterial SMK box riboswitch. RNase P is an essential enzyme present in all three domains of life, responsible for cleaving the 5' leader sequence of precursor tRNAs, yielding mature tRNAs. The most well characterized RNase P is the bacterial version, which was shown to be an RNA enzyme, or ribozyme. Although the RNase P RNA (RPR) is the catalytic moiety, the enzyme is also a ribonucleoprotein, containing a small RNase P protein (RPP) subunit that binds to the large RNA and accounts for ~10% of the enzyme's total mass. The RPR is catalytically active in vitro under high salt conditions, but requires the RPP for activity in vivo. RNase P from eukaryotes is less well characterized. Its RPR is homologous to the bacterial RPR, yet no eukaryal protein has been found to share marked sequence similarity with the single bacterial RPR. Eukaryal RNase P contains at least nine protein subunits that account for ~70% the mass of the intact enzyme and whose roles in the enzyme's function remain unknown. Remarkably, the eukaryal RPR has only nominal enzymatic activity. We hypothesize that evolution has shifted structural or catalytic responsibilities from the RPR to the RPPs in the case of the eukaryal enzyme as compared to its bacterial counterpart. A subset of the eukaryal RPPs shares homology with four archaeal RPPs (POP5, RPP21, RPP29, and RPP30), which have been shown to enhance the activity of a cognate RPR in an in vitro reconstitution assay. We thus adopted archaeal RNase P as a model system, to gain insight into the eukaryal version of the enzyme by analogy. Archaeal RNase P is an attractive target for structural studies due to the demonstrated in vitro reconstitution, the limited number and smaller size of its RPPs, and the behavior of its thermophilic components. Work detailed here focuses primarily on the structure and interactions (open full item for complete abstract)

Committee: Mark Foster PhD (Advisor); Charles Bell PhD (Committee Member); Venkat Gopalan PhD (Committee Member); Thomas Magliery PhD (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, The Ohio State University, 2009, Microbiology

Organisms have evolved a variety of mechanisms for regulating gene expression. Expression of individual genes is carefully modulated during different stages of cell development and in response to changing environmental conditions. A number of regulatory mechanisms involve structural elements within messenger RNAs (mRNAs) that, in response to an environmental signal, undergo a conformational change that affects expression of a gene encoded on that mRNA. RNA elements of this type that operate independently of proteins or translating ribosomes are termed riboswitches. In this work, the THI box and SMK box riboswitches were investigated in order to gain insight into the structural basis for ligand recognition and the mechanism of regulation employed by each of these RNAs. Both riboswitches are predicted to regulate at the level of translation initiation using a mechanism in which the Shine-Dalgarno (SD) sequence is occluded in response to ligand binding. For the THI box riboswitch, the studies presented here demonstrated that 30S ribosomal subunit binding at the SD region decreases in the presence of thiamin pyrophosphate (TPP). Mutation of conserved residues in the ligand binding domain resulted in loss of TPP-dependent repression in vivo. Based on these experiments two classes of mutant phenotypes were identified. Class I mutations resulted in increased accessibility of the SD region and binding of 30S ribosomal subunits regardless of the presence or absence of TPP. In contrast, Class II mutations resulted in constitutive occlusion of the SD region even in the absence of ligand. The latter class represents the first example of riboswitch mutations that result in stabilization of the ligand-bound conformation when no ligand is present. For the SMK box riboswitch, mutational analysis verified the importance of conserved residues for binding to S-adenosylmethionine (SAM). A minimal SMK box element that retains the ability to bind SAM was used to determine the high resol (open full item for complete abstract)

Committee: Tina Henkin PhD (Advisor); Ross Dalbey PhD (Committee Member); Kurt Fredrick PhD (Committee Member); Mike Ibba PhD (Committee Member) Subjects: Biochemistry; Molecular Biology