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The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro
Author(s) -
Reich Christoph,
Zeller Mirijam,
Milkereit Philipp,
Hausner Winfried,
Cramer Patrick,
Tschochner Herbert,
Thomm Michael
Publication year - 2009
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2008.06577.x
Subject(s) - biology , specificity factor , rna polymerase , polymerase , rna polymerase ii , protein subunit , transcription factor ii d , rna polymerase i , microbiology and biotechnology , transcription (linguistics) , biochemistry , rna dependent rna polymerase , rna , enzyme , promoter , gene , gene expression , linguistics , philosophy
Summary The general subunit of all three eukaryotic RNA polymerases, Rpb12, and subunit P of the archaeal enzyme show sequence similarities in their N‐terminal zinc ribbon and some highly conserved residues in the C‐terminus. We report here that archaeal subunit P under the control of a strong yeast promoter could complement the lethal phenotype of a RPB12 deletion mutant and that subunit Rpb12 from yeast can functionally replace subunit P during reconstitution of the archaeal RNA polymerase. The ΔP enzyme is unable to form stable open complexes, but can efficiently extend a dinucleotide on a premelted template or RNA on an elongation scaffold. This suggests that subunit P is directly or indirectly involved in promoter opening. The activity of the ΔP enzyme can be rescued by the addition of Rpb12 or subunit P to transcription reactions. Mutation of cysteine residues in the zinc ribbon impair the activity of the enzyme in several assays and this mutated form of P is rapidly replaced by wild‐type P in transcription reactions. The conserved zinc ribbon in the N‐terminus seems to be important for proper interaction of the complete subunit with other RNA polymerase subunits and a 17‐amino‐acid C‐terminal peptide is sufficient to support all basic RNA polymerase functions in vitro .