Functional redundancy between the C-terminal domains of spt5 and rpb1 in Schizosaccharomyces pombe

Versatile mechanisms that control the coupling of essential modifications of the nascent transcript to transcription elongation are still poorly understood. My thesis focuses on the interplay between RNA polymerase II and transcription elongation factor Spt5 in Schizosaccharomyces pombe.;Spt5 exists in a complex with its co-factor Spt4. The Spt5-Spt4 complex is involved in the promoter-proximal pausing of RNA polymerase II transcription complexes. Phosphorylation of Spt5 by P-TEFb is required to release the stalled RNA polymerase II complex.;In fission yeast the C-terminal domain (CTD) of Spt5 spans amino acids 801-990 and harbors 18 nonamer repeats with the consensus sequence 1 TPAWNSGSK9. A model in which the C-terminal domain (CTD) of Spt5 functions as a docking platform for binding of processing enzymes, similarly to the CTD of Rpb1 as part of a transcription elongation checkpoint, has been proposed. Specifically, capping enzymes were shown to interact with both, the CTD of Spt5 and the CTD of Rpb1.;I set out to assess the function of the CTD of Spt5 for viability in fission yeast. I showed that the C-terminal domain of Spt5 is required to support normal growth. In its absence the cells grow slowly and exhibit elongated cell morphology. I identified residues within 1TPAWNSGSK9 that are important for interaction with the capping enzymes Pct1 and Pce1 and for phosphorylation by Cdk9/Pch1 in vitro. Furthermore I showed the impact of alanine substitutions of residues in 1TPAWNSGSK 9 on the growth phenotype of S. pombe in vivo.;In addition, I crossed truncated variants of rpb1 to truncated variants of spt5 or to spt5 mutants that were defective for interaction with the capping enzymes and/or for phosphorylation by Cdk9/Pch1. The results revealed that the CTDs of rpb1 and spt5 are functionally redundant.;Spt4 is the co-factor of Spt5. I demonstrated that while Spt4 is not required for viability, it gains importance in supporting growth of S. pombe in the absence of the C-terminal domain in Spt5.