Desulfovibrio vulgaris Hildenborough (DvH) is a sulfate-reducing bacterium that has the potential to be an agent of bioremediation by aiding in the precipitation of heavy metals and radionuclides from contaminated environments via its natural metabolism. Because such environments are commonly associated with a variety of bacterial stressors, it is important to understand how Desulfovibrio regulates its response to changing conditions for successful environmental application. Very few types of regulation have been characterized in Desulfovibrio and many regulatory networks remain unknown. The importance of regulation by both cis and trans acting RNA molecules has become increasingly evident over the last decade. Both small RNAs and riboswitches have been shown in other bacteria to play key roles in sensing cellular conditions and eliciting the appropriate responses. A previous study in this laboratory identified several small regulatory RNAs, but their biological roles have remained largely unknown. Additionally, a few uncharacterized riboswitches have been predicted in DvH based on genome sequence analysis. This dissertation aimed to characterize the most conserved DvH sRNA and to investigate its potential dual role as a S-adenosylmethionine (SAM) sensing riboswitch. Expression of the sRNA, Dv SIC19, was confirmed during normal growth in lactate/sulfate medium via Northern blot analysis. Sequence and expression analyses also indicated that Dv SIC19 is located upstream of a small hypothetical protein DVU1170 and that the two genes are co-transcribed. Molecular techniques and computational analysis were also employed to determine a role for Dv SIC19 and to identify potential targets. Stress analysis using qRT-PCR suggested a potential role for Dv SIC19 under normal growth conditions as opposed to being involved in a specific stress response. While characterization of Dv SIC19 was ongoing, re-annotation of the DvH genome indicated that Dv SIC19 shared both sequence and structural similarity to the SAM-I class of riboswitches. Multiple techniques, both in vitro and in vivo were used to verify the riboswitch activity of Dv SIC19 and its response to SAM. Determining that Dv SIC19 played some role in the methionine biosynthesis pathway lead to the identification of a mRNA target encoding SahR, a predicted transcriptional regulator of methionine biosynthesis genes. Subsequent electrophoretic mobility shift assays confirmed the ability of Dv SIC19 to bind the sahR transcript and qRT-PCR analysis of a Dv SIC19 deletion strain suggested a negative regulatory role. This study presented the first regulatory role for a newly discovered sRNA in Desulfovibrio. Additionally, this study verified that Dv SIC19 acts not only as a trans regulatory molecule, but also as a cis regulatory element in the methionine biosynthesis pathway of DvH.

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Orientador : Profª. Drª. Maria Berenice R. Steffens / Coorientador : Profª. Drª. Michelle Zibetti Tadra Sfeir / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências : Bioquímica. Defesa: Curitiba, 29/09/2016 / Inclui referências : f. 89-116 / Resumo: Herbapirillum seropedicae SmR1 é uma bactéria diazotrófica que coloniza, de forma endofítica, várias plantas de interesse econômico. A identificação e caracterização de RNAs não codificadores (ncRNAs) no gênero Herbaspirillum é uma etapa importante para o estudo da interação dessas moléculas com mRNAs- ou proteínas-alvo, no processo de regulação pós-transcricional. Neste trabalho foi ampliou-se a análise estrutural e funcional de ncRNAs com função regulatória em H. seropedicae SmR1. Além disso, foram preditos ncRNAs em outras bactérias do gênero Herbaspirillum. Utilizando a ferramenta Infernal 1.1.1 foram identificados 55 novos ncRNAs H. seropedicae SmR1, com expressão confirmada, que foram somados aos 173 já relatados por Moreno (2013) e Cirino (2014). Os novos ncRNAs foram classificados de acordo com a sua estrutura, como ncRNA cis-encoded ou ncRNAs trans-encoded. Foram também encontrados riboswitches e um representante da nova classe de RNA, a sequência CRISPR. Utilizando a ferramenta TargetRNA2 foram preditos diversos mRNAs-alvos. Dez ncRNAs de H. seropeddicae SmR1 com expressão confirmada em experimentos de RNAseq foram selecionados e oligonucleotídeos foram desenhados para futura validação por qRT-PCR. Finalmente, estes resultados contribuirão para o entendimento da participação desse tipo de RNA na regulação do metabolismo de bactérias do gênero Herbaspirillum. Palavras-chave: H. seropedicae SmR1; RNA não codificador, ncRNA, cis-encoded ncRNA, trans-encoded ncRNA, riboswitch, CRISPR. / Abstract: Herbaspirillum seropedicae SmR1 is a diazotrophic bacterium that colonizes endophytically several plants of economic interest. The identification and characterization of non-coding RNAs (ncRNAs) in the genus Herbaspirillum is an important step in the study of the interaction of these molecules with mRNAs- or proteins-tragets, in the post-transcriptional regulation process. In this work we continued the study of the structural and functional analysis of ncRNAs with regulatory function in H. seropedicae SmR1. In addition, new ncRNAs were predicted in other bacteria of the genus Herbaspirillum. Using the Infernal 1.1.1 tool, 55 new ncRNAs, with confirmed expression, were identified in the H. seropedicae SmR1 genome. They were added to the 173 ncRNas already reported by Moreno (2013) and Cirino (2014). The new ncRNAs were classified as cis-encoded ncRNAs or trans-encoded ncRNAs. We also found riboswitches and a representative of the new RNA class, the CRISPR sequence. Using the TargetRNA2 tool, several mRNAs-target were predicted. Ten ncRNAs of H. seropedicae SmR1, with confirmed expression in RNAseq experiments, were selected and oligonucleotides were designed for further validation by qRT-PCR. Finally, these results will contribute to the understanding of the participation of this type of RNA in the regulation of the metabolism of bacteria of the genus Herbaspirillum. Key-words: H. seropedicae SmR1; RNA non-coding (ncRNA), ncRNA cis_encoded, trans-encoded, riboswitches, CRISPR, mRNA.

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Riboswitches are cis-acting gene regulatory RNAs, which function without involvement of proteins. They have been implicated as drug targets and are attractive systems for the study of RNA-ligand binding and RNA folding. The purine riboswitch was used as a model system for RNA-ligand docking. Published binding data was successfully reproduced in silico and compounds predicted to bind the riboswitch in a virtual screening were tested experimentally. Structural data confirming the predicted binding mode for several cases was obtained. The problems encountered were not specific to RNA-ligand docking but known from the far more explored field of protein-ligand docking.The SAM-I riboswitch was also subjected to virtual ligand screening. This receptor is a system of greater complexity than the purine riboswitch and consequently posed a harder challenge to the docking protocol. After initial validation of the docking setup based on previously published data, a set of compounds selected from the in-house database of commercially available compounds was screened. One compound identfied in silico was cofirmed to bind experimentally.The k-turn motif found in the SAM-I riboswitch was investigated with respect to its folding. The k-turn motif was found to be foldable in context of the SAMI riboswitch as well as in isolation as was expected. Furthermore, mutations disrupting key interactions within the k-turn motif were found to be prohibitive of k-turn folding in isolation as well as in context of the riboswitch, leading to a loss of ligand binding. Interestingly, two sequences were identfied which fold in context of the riboswitch but do not fold in isolation. This confirms the contribution of tertiary interactions to k-turn folding. This conclusion was backed up with structural data is a system of greater complexity than the purine riboswitch and consequently posed a harder challenge to the docking protocol. After initial validation of the to its folding. The k-turn motif was found to be foldable in context of the SAMI riboswitch as well as in isolation as was expected. Furthermore, mutations disrupting key interactions within the k-turn motif were found to be prohibitive of k-turn folding in isolation as well as in context of the riboswitch, leading to a loss of ligand binding. Interestingly, two sequences were identi ed which fold in context of the riboswitch but do not fold in isolation. This con rms the contribution of tertiary interactions to k-turn folding. This conclusion was backed

Thesis advisor: Michelle M. Meyer / Non-coding RNA (ncRNA) perform vital functions in cells, but the impact of diversity across structure and function of homologous motifs has yet to be fully investigated. One reason for this is that the standard phylogenetic analysis used to address these questions in proteins cannot easily be applied to ncRNA due to their inherent characteristics. Compared to proteins, ncRNA have shorter sequence lengths, lower sequence conservation, and secondary structures that need to be incorporated into the analysis. This has necessitated an effort to develop methodology for investigating the evolutionary and functional relationship between sets of ncRNA. In this pursuit, I studied closely related riboswitches. Riboswitches are structured ncRNA found in bacterial mRNA that regulate gene expressions using their two major components: the aptamer and the expression platform. The aptamer of a riboswitch is able to bind a specific small molecule (ligand), and the bound/unbound state of the aptamer influences conformational changes in the expressions platform that can lead to increased or decreased downstream gene expression. Utilizing sequence and structural similarity metrics combined with graph clustering and de novo community detection algorithms I have determined a methodology for investigating the functional and evolutionary relationship between closely related riboswitches, and other ncRNA by extension, that are found across a range of diverse phyla. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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Thesis advisor: Michelle M. Meyer / Bacteria commonly utilize cis-acting mRNA structures that bind specific molecules to control gene expression in response to changing cellular conditions. Examples of these ligand-sensing RNA cis-regulators are found throughout the bacterial world and include riboswitches, which interact with small metabolites to modulate the expression of fundamental metabolic genes, and the RNA structures that bind select ribosomal proteins to regulate entire ribosomal protein operons. Despite advances in both non-coding RNA discovery and validation, many predicted regulatory RNA motifs remain uncharacterized and little work has examined how RNA cis-regulators behave within their physiological context in the cell. Furthermore, it is not well understood how structured RNA regulators emerge and are maintained within bacterial genomes. In this thesis, I validate the biological function of a conserved RNA cis-regulator of ribosomal protein synthesis previously discovered by my group using bioinformatic approaches. I then investigate how bacteria respond to the loss of two different cis-regulatory RNA structures. Using Bacillus subtilis as a model organism, I introduce point mutations into the native loci of the ribosomal protein L20-interacting RNA cis-regulator and the tandem glycine riboswitch and assay the strains for fitness defects. I find that disrupting these regulatory RNA structures results in severe mutant phenotypes, especially under harsh conditions such as low temperatures or high glycine concentrations. Together, this body of work highlights the advantages of examining RNA behavior within its biological context and emphasizes the important role RNA cis-regulators play in overall organismal viability. My studies shed light on the selective pressures that impact structured RNA evolution in vivo and reinforce the potential of cis-regulatory RNAs as novel antimicrobial targets. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Riboswitches are gene regulatory elements found in messenger RNA that function by changing structure upon the binding of a ligand to an aptamer domain. Single adenine-binding pbuE riboswitch aptamer RNAs were unfolded and refolded co-transcriptionally using optical tweezers for single molecule force spectroscopy. The kinetic and energetic properties of distinct folding intermediates were characterised with and without the binding of adenine. These observed intermediates were related to structural elements of the aptamer, which were found to fold sequentially, in a transcriptionally independent manner. The mechanical switch underlying the regulatory action of the riboswitch was observed directly (adenine stabilisation of the weakest helix), and the energy landscape for the folding was reconstructed. The construction of a dual-beam optical trap with separate detection and trapping laser beams manipulated and focused into a rigid, modified inverted microscope is also described. This instrument aims to achieve ngstrm-level resolution through careful design to reduce noise.