The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro.
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 DeltaP 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 DeltaP 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.
Reich
Christoph
C
Lehrstuhl für Mikrobiologie, Universitat Regensburg, Regensburg, Germany.
Zeller
Mirijam
M
Milkereit
Philipp
P
Hausner
Winfried
W
Cramer
Patrick
P
Tschochner
Herbert
H
Thomm
Michael
M
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D021122:Protein Subunits; D012321:DNA-Directed RNA Polymerases