Project description:Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. Our project is to show that the conserved RNA-binding Seb1 is a pausing-promoting factor for RNA polymerases I to control cotranscriptional RNA processing. Here, we did a proximity dependent biotinylation followed by mass spectrometry (PDB-MS) of the Seb1 protein in order to assess for physical interactions with the RNAPI transcription machinery. A mutant E. coli BirA enzyme is fused to the Seb1 protein. This mutant version of BirA uses biotin to catalyze the formation of biotinoyl-5′-AMP (bioAMP), thereby generating a ‘cloud’ of activated biotin molecules that can react with free primary amines (most often lysine residues) of neighboring proteins. This experiment will support the conclusion that Seb1 is located at the rDNA locus.
Project description:Hrp3_Purification from Schizosaccharomyces pombe 972h- Eukaryotic genome is composed of repeating units of nucleosomes to form chromatin arrays. A canonical gene is marked by nucleosome free region (NFR) at its 5’ end followed by uniformly spaced arrays of nucleosomes. In fission yeast we show both biochemically and in vivo that both Hrp1 and Hrp3 are key determinants of uniform spacing of genic arrays.
Project description:Interconnections between RNA-processing pathways revealed by a sequencing-based genetic screen for pre-mRNA splicing mutants in fission yeast
