Seb1 proximity dependent biotinylation (PDB) in Schizosaccharomyces pombe

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.

RNA聚合酶I（RNA polymerase I, RNAPI）介导的转录活动占真核细胞总转录活性的绝大多数，且与成熟核糖体RNA（ribosomal RNA, rRNA）的生成紧密关联。由于多项rRNA成熟步骤与RNAPI转录过程相偶联，RNAPI的延伸速率会直接调控新生前体rRNA的加工；而RNAPI转录速率的改变，可在应对生长条件变化与应激刺激时，触发替代性的rRNA加工通路。然而，目前通过调控转录延伸速率以控制RNAPI行进过程的相关因子与机制，仍未得到充分阐释。本研究证实，保守型RNA结合蛋白Seb1可作为RNA聚合酶I的转录暂停促进因子，进而调控共转录RNA加工过程。在此，我们将突变型大肠杆菌（E. coli）BirA酶与Seb1蛋白融合，对Seb1蛋白开展邻近依赖型生物素标记联合质谱（proximity dependent biotinylation followed by mass spectrometry, PDB-MS）分析，以探究其与RNAPI转录机器的物理相互作用。此突变型BirA可利用生物素催化生成生物素酰-5'-腺苷一磷酸（bioAMP），进而形成活化生物素分子的“活性云团”，该云团可与邻近蛋白的游离伯胺（多为赖氨酸残基）发生共价结合反应。本实验将为“Seb1定位于核糖体DNA（rDNA）基因座”这一结论提供实验支撑。