Site specific phosphorylation of yeast RNA polymerase I

Titelangaben

Gerber, Jochen ; Reiter, Alarich ; Steinbauer, Robert ; Jakob, Steffen ; Kuhn, Claus-D. ; Cramer, Patrick ; Griesenbeck, Joachim ; Milkereit, Philipp ; Tschochner, Herbert:
Site specific phosphorylation of yeast RNA polymerase I.
In: Nucleic Acids Research. Bd. 36 (2008) Heft 3 . - S. 793-802.
ISSN 1362-4962
DOI: https://doi.org/10.1093/nar/gkm1093

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Abstract

All nuclear RNA polymerases are phosphoprotein complexes. Yeast RNA polymerase I (Pol I) contains approximately 15 phosphate groups, distributed to 5 of the 14 subunits. Information about the function of the single phosphosites and their position in the primary, secondary and tertiary structure is lacking. We used a rapid and efficient way to purify yeast RNA Pol I to determine 13 phosphoserines and -threonines. Seven of these phosphoresidues could be located in the 3D-homology model for Pol I, five of them are more at the surface. The single phosphorylated residues were systematically mutated and the resulting strains and Pol I preparations were analyzed in cellular growth, Pol I composition, stability and genetic interaction with non-essential components of the transcription machinery. Surprisingly, all Pol I phosphorylations analyzed were found to be non-essential post-translational modifications. However, one mutation (subunit A190 S685D) led to higher growth rates in the presence of 6AU or under environmental stress conditions, and was synthetically lethal with a deletion of the Pol I subunit A12.2, suggesting a role in RNA cleavage/elongation or termination. Our results suggest that individual major or constitutively phosphorylated residues contribute to non-essential Pol I-functions.