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Schizosaccharomyces pombe

ABSTRACT: Genome-wide binding analysis of transcription factors regulating the expression of ribosomal-related genes.

PROVIDER: PRJDB16685 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	DRR505088_1.fastq.gz	Fastqsanger.gz
	DRR505088_2.fastq.gz	Fastqsanger.gz
	DRR505089_1.fastq.gz	Fastqsanger.gz
	DRR505089_2.fastq.gz	Fastqsanger.gz
	DRR505090_1.fastq.gz	Fastqsanger.gz

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Seb1 proximity dependent biotinylation (PDB) in Schizosaccharomyces pombe

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.

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Project description:ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including S. pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.

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