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Molecular and Cellular Biology, December 2000, p. 9307-9316, Vol. 20, No. 24
Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston,
Massachusetts 02115
Received 26 July 2000/Returned for modification 7 September
2000/Accepted 27 September 2000
The Saccharomyces cerevisiae mRNA capping enzyme
consists of two subunits: an RNA 5'-triphosphatase (Cet1) and an mRNA
guanylyltransferase (Ceg1). In yeast, the capping enzyme is recruited
to the RNA polymerase II (Pol II) transcription complex via an
interaction between Ceg1 and the phosphorylated carboxy-terminal domain
of the Pol II largest subunit. Previous in vitro experiments showed
that the Cet1 carboxy-terminal region (amino acids 265 to 549) carries
RNA triphosphatase activity, while the region containing amino acids
205 to 265 of Cet1 has two functions: it mediates dimerization with
Ceg1, but it also allosterically activates Ceg1 guanylyltransferase
activity in the context of Pol II binding. Here we characterize several
Cet1 mutants in vivo. Mutations or deletions of Cet1 that disrupt
interaction with Ceg1 are lethal, showing that this interaction is
essential for proper capping enzyme function in vivo. Remarkably, the
interaction region of Ceg1 becomes completely dispensable when Ceg1 is
substituted by the mouse guanylyltransferase, which does not require
allosteric activation by Cet1. Although no interaction between Cet1 and
mouse guanylyltransferase is detectable, both proteins are present at yeast promoters in vivo. These results strongly suggest that the primary physiological role of the Ceg1-Cet1 interaction is to allosterically activate Ceg1, rather than to recruit Cet1 to the Pol II complex.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
The Essential Interaction between Yeast mRNA
Capping Enzyme Subunits Is Not Required for Triphosphatase Function
In Vivo

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Corresponding author. Mailing address: Department of
Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, MA 02115. Phone: (617) 432-0696. Fax: (617) 738-0516. E-mail: steveb{at}hms.harvard.edu.
Present address: Eisai Tsukuba Research Laboratories, Ibaraki
300-26, Japan.
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