Petrakis, T.G.; (2005) Architecture and interactions of the Saccharomyces cerevisiae elongator complex. Doctoral thesis , University of London.

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Abstract

Yeast Elongator was initially isolated based on its interaction with the hyper-phosphorylated form of RNA polymerase II. Later on, it was shown to possesion intrinsic histone acetyl-transferase activity and to crosslink to nascent RNA in vivo. The six ELP genes were also identified, among other genes, in a genetic screen in Saccharomyces cerevisiae for targets of the toxin zymocin. KTI12, which stands for *jC lactis toxin Insensitive 12", was one of those other genes. ktil2A mutant cells display phenotypes closely resembling those of elpA mutants. Moreover, Ktil2 protein was shown to co-immunoprecipitate with Elongator and with RNA polymerase II, indicating a functional interaction with both factors. The experiments presented in the first part of this thesis confirm genetically and biochemically that Elongator is a six-subunit complex. In vitro and in vivo studies were performed to reveal the molecular architecture of this complex. Briefly, strong pair-wise interactions between Elpl and Elp3, Elp4 and Elp6 and between Elp5 and Elp6 were uncovered. Additionally, a weak interaction between Elp3 and Elp4 was observed. In vitro HAT assays and RNA immunoprecipitation experiments suggested that Elp2 is dispensable for the in vitro histone acetyl-transferase and the in vivo RNA binding activity of Elongator. In contrast, Elp3 was shown to be critical for both the integrity of the complex and its in vivo RNA binding activity. Moreover, the localisation of yeast Elp3 protein was studied, in an attempt to address the possibility that Elongator continuously shuttles from the cytoplasm to the nucleus. In the second part, biochemical and genetic studies strongly suggested that Ktil2 interacts with Elongator and might regulate its in vivo HAT activity. Finally, the molecular mechanism of action of the toxin zymocin was studied. In particular, preliminary experiments, which test the possibility that the RNAPII gets degraded in response to zymocin treatment of 5. cerevisiae cells, are presented.

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