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J Biol Chem, Vol. 275, Issue 18, 13780-13788, May 5, 2000
From the Banting and Best Department of Medical Research,
Toronto, Ontario M5G 1L6, Canada
Atomic absorption spectroscopy demonstrated that
highly purified RNA polymerase II from the yeast Saccharomyces
cerevisiae binds seven zinc ions. This number agrees with the
number of potential zinc-binding sites among the 12 different subunits
of the enzyme and with our observation that the ninth largest subunit
alone is able to bind two zinc ions. The zinc-binding motif in the
largest subunit of the enzyme was investigated using mutagenic
analysis. Altering any one of the six conserved residues in the
zinc-binding motif conferred either a lethal or conditional phenotype,
and zinc blot analysis indicated that mutant forms of the domain had a
2-fold reduction in zinc affinity. Mutations in the zinc-binding domain
reduced RNA polymerase II activity in cell-free extracts, even though
protein blot analysis indicated that the mutant subunit was present in
excess of wild-type levels. Purification of one mutant RNA polymerase
revealed a subunit profile that was wild-type like with the exception
of two subunits not required for core enzyme activity (Rpb4p and
Rpb7p), which were missing. Core activity of the mutant enzyme was
reduced 20-fold. We conclude that mutations in the zinc-binding domain
can reduce core activity without altering the association of any of the
subunits required for this activity.
Zinc Stoichiometry of Yeast RNA Polymerase II and
Characterization of Mutations in the Zinc-binding Domain of the
Largest Subunit*
and
*
This work was supported by a scholarship from the National
Science and Engineering Research Council (to I. M. D.) and Medical Research Council Grant MT-7912.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Current address: Samuel Lunenfeld Research Institute, Mount Sinai
Hospital, 600 University Ave., Toronto, Ontario M5G 1X5, Canada.
§
To whom correspondence should be addressed: Banting and Best Dept.
of Medical Research, 112 College St., Toronto, Ontario M5G 1L6, Canada.
Tel.: 416-946-3016; Fax: 416-978-8528; E-mail: james.friesen@
utoronto.ca.
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