Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Dissecting the functions of mycobacteria RbpA structural domains

ABSTRACT: RNA polymerase (RNAP) binding protein RbpA contributes to the formation of stable RNAP-promoter open complexes (RPo) and is essential for viability in mycobacteria. Based on structural and biochemical data, four domains have been identified in the RbpA protein: a N-terminal tail (NTT) domain of unknown function, a core domain (CD) that contacts the RNAP β’ subunit in a recently solved crystal structure, a basic linker (BL) that binds DNA, and a s-interaction domain (SID) that binds group I and group II s-factors. However, limited in vivo studies have been performed in mycobacteria and how the individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We dissected the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis. We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP holoenzyme, indicating that the primary function of the CD is not solely association with the RNAP. We show that RbpA BL and SID are required for stabilization of RPo complexes at the ribosomal RNA rrnAP3 promoter in vitro, while the NTT and CD are dispensable. Finally, we determine that the NTT and CD impact gene expression of a distinct set of genes from that affected by the BL and SID activities. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA.

ORGANISM(S): Mycolicibacterium smegmatis

PROVIDER: GSE107123 | GEO | 2018/05/03

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

Dissecting the functions of mycobacteria RbpA structural domains

Project description:Dissecting the functions of mycobacteria RbpA structural domains

| PRJNA419075 | ENA

RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Mycobacterium smegmatis

Project description:6S RNA is a small RNA with specific secondary structure that associates with the complex of RNA polymerase (RNAP) and the primary sigma factor in majority of bacteria. In mycobacteria, Ms1 interacts with the RNAP core without the sigma factor and probably replaces 6S RNA. To identify RNAs that have a similar function as Ms1 or to identify a new 6S RNA in mycobacteria, we sequenced RNAs that co-immunoprecipitated with RNAP or the primary sigma factor sigma A. We also sequenced total RNA isolated from the lysate (input sample). We expect that putative regulatory RNAs are enriched in RNA polymerase or sigma A samples compared to the inputs. The experiment was performed in exponential and stationary phase of growth.

2024-02-09 | E-MTAB-11692 | biostudies-arrayexpress

Arabidopsis thylakoid domains proteome

Project description:The thylakoid membrane in land plants is organized into an appressed domain (grana), enriched in photosystem (PS) II, and a non-appressed domain (stroma lamellae) enriched in PSI. This ultrastructure ensures control over excitons spillover from PSII to PSI. The bulky machinery required for the repair and biogenesis of PSII and other proteins of the stacked, appressed domain is located in the non-appressed membranes. Thus, the connecting domain (CD) between grana and stroma lamellae, also known as the grana margins (GM), is the key player in both the structural and functional integrity of the photosynthetic machinery. To elucidate the properties and composition of the GM proteome, we have analyzed the different thylakoid domains isolated from membranes in native conformation as well as from thylakoids artificially unstacked to induce a homogeneous protein composition. We show that the composition of the CD is strictly distinct from that of the highly curved structures (the curvature) present at the periphery of grana discs, which are not physically connected to the stroma lamellae. Furthermore, a detailed map of the composition, distribution and interaction of the protein complexes involved in thylakoid membrane function is provided for the grana, CD, curvature and stroma lamellae domains.

2025-02-11 | PXD046544 | Pride

Targeting the SIN3A-PF1 Interaction inhibits Epithelial to Mesenchymal Transition and Maintenance of a Stem Cell Phenotype in Triple Negative Breast Cancer (Expression)

Project description:Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial to mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting. Sub-confluent cultures of MDA-MB-231 cells were treated with 1ÂµM Tat-SID peptide or 1ÂµM Tat-Scr scrambled control peptide for 24hr.

2015-11-02 | E-GEOD-73278 | biostudies-arrayexpress

Targeting the SIN3A-PF1 Interaction inhibits Epithelial to Mesenchymal Transition and Maintenance of a Stem Cell Phenotype in Triple Negative Breast Cancer (MNase-seq)

Project description:Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial to mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting. Mononucleosomes from MDA-MB-231 cells were isolated and ChIP with H3K4me3 antibody. DNA from Input and ChIP samples was purified and sequenced on Illumina Hiseq.

2015-11-02 | E-GEOD-73869 | biostudies-arrayexpress

MoaB2 binds to SigA in Mycobacterium smegmatis and modulates its activity and biological stability

Project description:In mycobacteria, SigA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and is responsible for initiating transcription of housekeeping genes. Our knowledge about this factor in medicinally important mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis SigA. The search revealed a number of proteins; prominent among them was MoaB2. The SigA-MoaB2 interaction was validated and characterized by several approaches, revealing that it does not require RNAP and is highly specific, as alternative sigma factors did not interact with MoaB2. The structure of MoaB2 was solved by crystallography and the unique, unstructured N-terminal domain of SigA was identified to play a role in the SigA-MoaB2 interaction. Functional experiments showed that MoaB2 inhibits SigA-dependent transcription and increases biological stability of SigA. We propose that MoaB2, by sequestering SigA, exerts a dual effect on the transcription apparatus, and has the potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial SigA, paving the way for future research of this phenomenon.

2024-11-11 | PXD039468 | Pride

The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs

Project description:Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here we examined the contribution of structural domains in Piwi proteins towards defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5′ uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5’ end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-PIWI module of a Piwi protein in dictating the strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis

2014-07-01 | GSE55451 | GEO

The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs

Project description:Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here we examined the contribution of structural domains in Piwi proteins towards defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5M-bM-^@M-2 uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5M-bM-^@M-^Y end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-PIWI module of a Piwi protein in dictating the strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis Immunoprecipitated small RNA were purified from Bmn4 cells for preparation of high-throughput sequencing libraries.

2014-07-01 | E-GEOD-55451 | biostudies-arrayexpress