Regulation Of Transcription By RNA Polymerase II In S. pombe

Gopalan, Sneha (2018). Regulation Of Transcription By RNA Polymerase II In S. pombe. PhD thesis The Open University.



An understanding of the mechanisms underlying the various stages of transcription is crucial to find solutions to the problems caused by mis-expression of genes that may give rise to a host of human diseases. My thesis research focusses on an analysis of the RNA Polymerase II elongation factor Ell1/Eaf1 in S. pombe. Eleven-nineteen lysine-rich in leukemia (ELL) is encoded by a gene involved in translocations with MLL in leukemia and forms a tight complex with ELL-associated factors (EAF). ELL/EAF is an RNA polymerase II elongation factor that in metazoa can assemble into a larger assembly that also includes P-TEFb and other proteins encoded by genes involved in MLL translocations. This larger assembly, sometimes called SEC, binds to a specific "docking site" in the metazoan Mediator complex. Distantly related ELL- and EAF-like genes were identified in S. pombe that encode Ell1/Eaf1 and can stimulate Pol II elongation in vitro. My thesis addresses two distinct projects, with overlapping motivations:

First, to see whether S. pombe might provide a good model for functional studies of the Mediator and ELL/EAF interaction, I carried out a thorough proteomic analysis of S. pombe Mediator and defined several new subunits. My results were recently published as part of a collaborative structural analysis of S. pombe Mediator.

Second, I used a combination of biochemical, genetic, and genomic approaches to characterize Ell1/Eaf1 function in fission yeast. Using mass spectrometry, I identified an uncharacterized sequence orphan, SPAC6G9.15c, that associates with both Ell1 and Eaf1 to form a ternary complex that, based on ChIP-seq localizes at genes with high RNA Pol II occupancy. I also performed an SGA screen for genes that genetically interact with ell1, eaf1, and SPAC6G9.15c and identified a set of overlapping genes that interact with all three, as well as others that interact only with ell1.

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