This thesis explores the RNA folding problem using single-molecule field effect transistors (smFETs) to measure the lifetimes of individual RNA base-pairing rearrangements. In the course of this research, considerable computational, chemical, and engineering contributions were developed so that the single-molecule measurements could be conducted and quantified. These advancements have allowed, on the basis of the smFET data collected herein, the quantification of a kinetic model for RNA stem-loop structures which has been generalized to quantitatively explore the phenomenological observation that an RNA found in the bacillus subtilis strain acts as a metabolite-sensing switch, allowing RNA polymerase to transcribe the messenger RNA when the metabolite is present and preventing transcription when the metabolite is absent. Together, the data presented quantify a simple model for the base pairing rearrangements that underlie RNA folding.

The 5’ untranslated region (UTR) of coronaviral genomes contains cis-acting sequences necessary for replication, transcription and translation. A consensus secondary structural model of the 5' 140 nucleotides of the 5' UTRs of nine coronaviruses (CoVs) derived from all three major CoV groups is presented and characterized by three major stem loops, SL1, SL2 and SL4. SL2 is conserved in all CoVs, typically containing a pentaloop (C47-U48-U49-G50-U51 in MHV) stacked on a 5-bp stem, with some sequences containing an additional U 3' to U51. NMR structural studies of SL2 hairpin reveal that SL2 adopts a U-turn-like conformation. Parallel molecular genetic experiments reveal that SL2 plays an essential role in sgRNA synthesis as does SL1. We observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a collection of non-canonical base pairs near the deleted A35. Viruses containing destabilizing SL1-∆A35 mutations also contain one of two specific single nucleotide mutations in the 3' UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-∆A35 substitutions recover one or more features of the wild-type SL1. We propose a "dynamic SL1" model that supports viral replication; these characteristics of SL1 appear to be conserved in other coronaviral genomes. The coronaviral nucleocapsid (N) protein contains two or more RNA binding domains. We investigated the RNA-binding properties of the N-terminal (NTD) and Cterminal (CTD) domain of MHV N. Our results reveal that the NTD specifically interacts with the TRS-L3 sequence. The role of conserved residues (Y127, Y129 and R110) for this specific interaction were systematically investigated. In contrast to the NTD, the MHV CTD is homodimeric in solution and binds single-strand RNA nonspecifically in a binding mode of the noncooperative large ligand lattice model. The CTD dimer binds with a site size, n=4 nucleotide and the appending of the NTD enhances the single-strand nucleic acid binding affinity.

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

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Work over the past two decades has revealed that non-coding RNAs (ncRNAs) are prevalent in all kingdoms of life. Using RNA-seq we discovered hundreds of ncRNAs in the antibiotic-producing genus of bacteria, Streptomyces. These included trans-encoded small RNAS (sRNAs), cis-antisense RNAs, and a new type of antisense RNA we termed cutoRNAs (convergent untranslated overlapping RNAs) that arise when transcription termination does not occur in the intergenic region between two convergently arranged genes. Many of these ncRNAs feature prominently in the specialized metabolite biosynthetic clusters (e.g. antibiotics, anticancer agents, immunosuppressants). Hence, it is likely that understanding the functions of these RNAs will be important for new molecule discovery. We found that one highly expressed antisense RNA (ScbN) was expressed opposite the -butyrolactone synthase scbA in the model streptomycete Streptomyces coelicolor. However, ScbN had no detectible impact on the expression of scbA. Instead, the transcription terminator of scbN, which also forms a hairpin within the coding sequence of scbA, was found to reduce expression of scbA more than 10-fold. This led us to bioinformatically search for similar coding-sequence hairpins throughout all bacteria, leading to the discovery of many stable RNA structures with conserved locations throughout very divergent bacteria (e.g. Streptomyces, Escherichia coli, Bacillus subtilis). / Thesis / Doctor of Philosophy (PhD) / The flow of genetic information, from DNA to RNA to proteins, often portrays RNA as a mere intermediary molecule. An alternative, and perhaps more accurate, way to view RNA is that it is central to all cellular processes. Many RNAs are not translated into proteins and instead act as regulatory molecules, impacting the expression of other genes. In this work we found many examples of these regulatory RNAs in a group of bacteria known to produce many of the world’s antibiotics. Understanding the roles these regulatory RNAs play in impacting gene expression will be important for the discovery of new molecules, such as antibiotics. In addition to distinct regulatory RNAs mentioned above, we found that RNA structures within the coding sequences of mRNAs that are translated into proteins have dramatic regulatory consequences. We describe the characterization of one such RNA structure in a gene involved in bacterial communication, and develop a bioinformatic tool to hunt for other such structures conserved throughout bacteria.

Genome-scale ordered RNA structure (GORS) was first identified in 2004. It refers to the presence of secondary structure throughout the length of the RNA genomes of certain genera of RNA virus families, as predicted by bioinformatic analysis. It was also observed that the viruses containing GORS were able to establish persistent infections in their natural hosts, raising the possibility that the presence of GORS could play a role in viral avoidance of the innate immune system. This thesis describes the first study of GORS and its possible role in persistence. Two GORS viruses have been studied, equine rhinitis A virus (ERAV) and murine norovirus (MNV). A 55% seroprevalence of ERAV has been determined in a cohort of Scottish horses indicating a wide exposure to the virus. Equine faecal samples were screened for ERAV by PCR with the intention of identifying a virus, possibly from a persistently infected animal, which would not have undergone any cell culture adaptations as laboratory strains have. Newly identified viruses would then be sequenced, their secondary structures predicted and further studies carried out. Unfortunately, none of the 50 faecal samples screened were positive and clinical isolates of ERAV provided by the Animal Health Trust were sequenced but were identical to laboratory strains, so the study then focussed on MNV. Prevalence of MNV in laboratory mice was determined by PCR of faecal samples to be 67%. MNV was also discovered in the faeces of a pet shop mouse and a wild wood mouse (Apodemus sylvaticus). The complete genomes of 4 laboratory mouse MNVs, the pet shop mouse and wood mouse MNVs were sequenced. Phylogenetic analysis showed the wood mouse MNV had a p distance of 23% from other MNVs, although the laboratory mice and pet shop mouse were closely related to other MNVs. Structural analysis of the genomes of 6 sequenced MNVs, including the wood mouse virus, showed all were GORS viruses. A laboratory strain of MNV, MNV-3, was serially passaged in RAW 264.7 cells to test the hypothesis that in an animal with an intact immune system, there is a pressure for GORS viruses to maintain their genomic RNA structure as a means of immune avoidance, and that cell culture adaptation would attenuate the degree of secondary structure. The complete genome of passage 33 was sequenced, which revealed 7 base mutations, a mutation rate of 0.1 %, which was not considered significant enough to have affected the degree of secondary structure. In order to assess if structured and unstructured RNA behaved differently in cells, replication deficient RNA transcripts were made from the infectious clones of a panel of GORS and non-GORS viruses. These transcripts were electroporated into cells and their rate of decay measured, but there was no difference between the GORS and non-GORS transcripts. The full length and 4 kilobase transcripts were transfected into NIH3T3 cells and the degree of interferon-β induction measured by quantitative PCR and a luciferase reporter assay. The IFN-β response differed across the panel of viruses, and although none of the GORS viruses induced strongly, the non-GORS viruses were variable in their ability to induce an IFN-β response, some inducing strongly, other not at all. This result indicates that during exposure of viral genomes in the cytoplasm during infection, GORS-virus RNAs are unlikely to induce an interferon response, possibly contributing to their ability to persist. It is unclear why some non-GORS-viruses failed to induce IFN and there are likely to be other contributory factors.