Site specific phosphorylation of yeast RNA polymerase I.
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.
Gerber
Jochen
J
Institut für Biochemie, Mikrobiologie und Genetik, Universität Regensburg, Munich, Germany.
Reiter
Alarich
A
Steinbauer
Robert
R
Jakob
Steffen
S
Kuhn
Claus-Dieter
CD
Cramer
Patrick
P
Griesenbeck
Joachim
J
Milkereit
Philipp
P
Tschochner
Herbert
H
D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D012318:RNA Polymerase I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology
D005656:Fungal Proteins; D010750:Phosphoproteins; D010769:Phosphothreonine; D010768:Phosphoserine; D012318:RNA Polymerase I