RNA-seq of Mycobacterium smegmatis MC2-155 with or without inducible overexpression of Mtb RpfB UTR with Atc
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ABSTRACT: RpfB is one of five resuscitation promoting factors in Mycobacterium tuberculosis. We previously published data indicating the 5' UTR of the gene contains a regulatory riboswitch with an unknown (at the time) ligand. We overexpressed the switch in Mycobacterium smegmatis (which does not contain the rpfB gene), reasoning that overexpression of the switch would s sequester the unknown ligand, giving rise to a phenotype that would help us identify it.
Project description:RNAs are often studied in non-native sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods - a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added post-probing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5′ and 3′ ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer construct but introduced non-native interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for each nucleotide in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is an alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to non-native sequence elements.
Project description:Mycobacterium tuberculosis is an intracellular human pathogen with the ability to resist and adapt to many adverse conditions it encounters upon infection. Among these, overcoming the production of nitric oxide by macrophages could be key for M. tuberculosis success. We have challenged M. tuberculosis with a sub-lethal concentration of nitric oxide and followed the transcriptomic response through RNA-seq for 48 hours.
Project description:In this study we have combined RNA-seq analysis of genome-wide transcriptional start sites with regular RNA-seq to study the transcriptional landscape of Mycobacterium tuberculosis during exponential culture and growth arrest using a starvation model where exponentially growing cells are incubated in PBS for 24 hours. Three independent biological replicates were used in this experiment.
Project description:RNA is a multifaceted biomolecule with numerous biological functions and can interact with small molecule metabolites as exemplified by riboswitches. Here, we profile the Escherichia Coli transcriptome on interactions with the metabolite Thiamine Monophosphate (TMP). We designed and synthesized a photoaffinity probe based on the scaffold of TMP and applied it to chemotranscriptomic profiling. Using next-generation RNA sequencing, several potential interactions between bacterial transcripts and the probe were identified. A remarkable interaction between the TMP probe and the well-characterized ribB riboswitch was validated by RT-qPCR, and further verified with competition assays. Localization of the photocrosslinked nucleotides using reverse transcription and docking predictions of the probe suggested binding to the riboswitch aptamer. After examining binding of unmodified TMP to the riboswitch using SHAPE, we found selective yet moderate binding interactions, potentially mediated by the phosphate group of TMP. Lastly, TMP appeared to enhance gene expression of a reporter gene that is under riboswitch control, while the natural ligand Flavin Mononucleotide (FMN) displayed an inhibitory effect, hinting at a potential biological role of TMP. This work showcases the possibility of chemotranscriptomic profiling to identify new RNA-small molecule interactions.
Project description:The experiments were carried out to map the ligand binding landscape of various DNA and RNA duplexed aptamer families. Duplexed Aptamer (DA) constructs were engineered from (i) natural and synthetic DNA and RNA aptamers and (i) synthetic oligonucleotide aptamer-complementary elements synthesized on custom DNA microarrays. The aptamers tested consist of the ATP DNA aptamer, the ATP RNA aptamer, the cocaine DNA aptamer, the human alpha-thrombin DNA aptamer, and the natural add riboswitch aptamer from the pathogenic bacteria Vibrio vulnificus. Each duplexed aptamer family consists of 1000's of synthetic constructs, each formed by hybridizing the aptamer with an aptamer-complementary element (ACE) - here, ACEs consisted of various DNA oligonucleotides synthesized as a custom DNA microarray.
Project description:The experiments were carried out to map the ligand binding landscape of various DNA and RNA duplexed aptamer families. Duplexed Aptamer (DA) constructs were engineered from (i) natural and synthetic DNA and RNA aptamers and (i) synthetic oligonucleotide aptamer-complementary elements synthesized on custom DNA microarrays. The aptamers tested consist of the ATP DNA aptamer, the ATP RNA aptamer, the cocaine DNA aptamer, the human alpha-thrombin DNA aptamer, and the natural add riboswitch aptamer from the pathogenic bacteria Vibrio vulnificus. Each duplexed aptamer family consists of 1000's of synthetic constructs, each formed by hybridizing the aptamer with an aptamer-complementary element (ACE) - here, ACEs consisted of various DNA oligonucleotides synthesized as a custom DNA microarray.
Project description:The experiments were carried out to map the ligand binding landscape of various DNA and RNA duplexed aptamer families. Duplexed Aptamer (DA) constructs were engineered from (i) natural and synthetic DNA and RNA aptamers and (i) synthetic oligonucleotide aptamer-complementary elements synthesized on custom DNA microarrays. The aptamers tested consist of the ATP DNA aptamer, the ATP RNA aptamer, the cocaine DNA aptamer, the human alpha-thrombin DNA aptamer, and the natural add riboswitch aptamer from the pathogenic bacteria Vibrio vulnificus. Each duplexed aptamer family consists of 1000's of synthetic constructs, each formed by hybridizing the aptamer with an aptamer-complementary element (ACE) - here, ACEs consisted of various DNA oligonucleotides synthesized as a custom DNA microarray.