Dataset Information

Bacillus subtilis RNase M5 cleaves dsRNA in two steps via a two Mg2+-ion dependent mechanism

ABSTRACT: Here, we present the crystal structure of the catalytic domain of M5 bound to two magnesium ions, which reveals the role of each catalytic site acidic residue in metal binding. We further use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the possibility of structural rearrangements that would allow cleavage of the 5’-strand, and use small-angle X-ray scattering (SAXS) to study the M5 binding to the pre-5S rRNA substrate. This study provides new details on the M5 mechanism used for cleavage of the dsRNA substrate, which proceeds via two metal ions and structural rearrangements of both M5 and the pre-5S rRNA substrate.

INSTRUMENT(S): SYNAPT G2-Si

ORGANISM(S): Geobacillus Stearothermophilus

SUBMITTER: Maxime BOURGUET

LAB HEAD: Sarah CIANFERANI

PROVIDER: PXD019202 | Pride | 2021-02-10

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Cluster_data_M5_vs_M5-L18-RNA.csv	Csv
	Dataset_M5-L18-RNA_state.zip	Other
	Dataset_M5_state.zip	Other
	Dataset_final_peptide.zip	Other
	HD_plots.pptx	Other

Items per page:

1 - 5 of 8

Similar Datasets

Project description:We determined genome-wide nucleosome occupancy in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell type and protein specific binding preferences of transcription factors to sites with either low (e.g. Myc, Klf4, Zfx) or high (e.g. Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome depleted regions around transcription start and termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to their CpG-content or histone methylation marks. Throughout the genome nucleosome occupancy was dependent on the presence of certain histone methylation or acetylation modifications. In addition, the average nucleosome-repeat length increased during differentiation by 5-7 base pairs, with local variations for specific genomic regions. Our results reveal regulatory mechanisms of cell differentiation acting through nucleosome repositioning. The Total RNA from ESCs, NPCs and MEFs was extracted by guanidinisothiocyanat/phenol extraction with the Trifast kit (Peqlab). Total RNA preparations were treated with DNase I, phenol/chloroform extracted and precipitated before further processing. RNAs were depleted of 5S, 5.8S, 18S and 28S rRNAs using the Human/Mouse/Rat Ribo-Zero rRNA Removal Kit (Epicentre) according to the manufacturer’s protocol. After rRNA depletion, RNAs were fragmented with a kit from Ambion. Libraries for Solexa sequencing were generated according to the standard Illumina protocol that comprised first strand cDNA synthesis, second strand cDNA synthesis, end repair, addition of a single A base, and adapter ligation. Sequencing was performed on the Illumina GAIIx (replicate 1) and Illumina HiSeq 2000 (replicate 2) platforms at the sequencing core facilities of the BioQuant in Heidelberg, Germany. RNA reads were aligned with TopHat. Further expression analysis was with the Genomatix software suite (Genomatix, Munich, Germany) and the Eldorado gene annotation. For each transcript a normalized expression value was calculated from the read distribution that accounts for the length differences using the program DEseq for the analysis of differential expression.

Dataset Information

Bacillus subtilis RNase M5 cleaves dsRNA in two steps via a two Mg2+-ion dependent mechanism

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets