Purification and characterization of an RNA polymerase II phosphatase from yeast

RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by 30,000-fold. The y...

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Bibliographic Details
Published in:The Journal of biological chemistry 1996-10, Vol.271 (40), p.24498-24504
Main Authors: Chambers, R.S. (University of California, Berkeley, CA.), Kane, C.M
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Chromatography, Ion Exchange
Electrophoresis, Polyacrylamide Gel
ESTERASAS
ESTERASE
FOSFORILACION
HeLa Cells
Humans
Phosphoprotein Phosphatases
Phosphoric Monoester Hydrolases - chemistry
Phosphoric Monoester Hydrolases - metabolism
PHOSPHORYLATION
RNA Polymerase II - metabolism
SACCHAROMYCES CEREVISIAE
Saccharomyces cerevisiae - enzymology
TRANSFERASAS
TRANSFERASE
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Summary:RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by 30,000-fold. The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa. The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells. These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid. Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases. Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical. The activity of the highly purified CTD phosphatase is stimulated 300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity. These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.271.40.24498