Genetic and Proteomic Analysis of Rna Polymerase Ii-Interacting Complexes Mediator and Integrator in the Ciliated Protozoan Tetrahymena Thermophila
thesisposted on 24.05.2021, 13:51 by Matthew D. R. Cadorin
In most eukaryotes, the largest subunit of RNAPII, Rpb1, contains a conserved carboxyterminal domain (CTD) containing a canonical structure of heptapeptide repeats. Two protein complexes of interest, Mediator and Integrator, are known to interact with this CTD in all eukaryotic models they have been described in to date. Recently, orthologs of Mediator and Integrator subunits have been identified within the ciliated protozoan Tetrahymena thermophila; one of the few eukaryotic lineages to lack a canonically organized CTD. To begin to characterize putative Mediator and Integrator complexes within T. thermophila, I engineered appropriate macronuclear tagging and knockout cassettes. Although the Tetrahymena MED31 ortholog was unable to rescue the slow growth phenotype of a yeast MED31 knockout, or co-purify with yeast Med8-TAP, I identified subunit Med3 as a member of the Med31 interactome in T. thermophila through tandem affinity purification coupled with mass spectrometry. I also targeted the Tetrahymena INTS6 locus for knockout as determined by colony PCR. If Mediator and Integrator exist in Tetrahymena despite its divergent CTD of Rpb1, perhaps these complexes have CTD-independent functions beyond what can be effectively studied using conventional model systems.