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J. Biol. Chem., Vol. 278, Issue 17, 14949-14955, April 25, 2003
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From the Department of Biochemistry and Molecular Biology,
University of New Hampshire, Durham, New Hampshire 03824
The CCR4 family proteins are 3'-5'-deadenylases
that function in the first step of the degradation of poly(A) mRNA.
Here we report the purification to homogeneity of the yeast CCR4
protein and the analysis of its substrate specificities. CCR4
deadenylated a 7N+23A substrate (seven nucleotides followed by 23 A
residues) in a distributive manner. Only small differences in CCR4
activity for different A length substrates were observed until only 1 A residue remained. Correspondingly, the Km for a
25N+2A substrate was found to be at least 20-fold lower than that for a
26N+1A substrate, although their Vmax values
differed by only 2-fold. In addition, the total length of the RNA was
found to contribute to CCR4 activity: up to 17 nucleotides (not
necessarily poly(A)) could be recognized by CCR4. Poly(U), poly(C), and
poly(G) were also found to be 12-30-fold better inhibitors of CCR4
compared with poly(A), supporting the observation that CCR4 contains a non-poly(A)-specific binding site. Surprisingly, even longer substrates (
Identification of Multiple RNA Features That Influence CCR4
Deadenylation Activity*
45 nucleotides) stimulated CCR4 to become a processive enzyme, suggesting that CCR4 undergoes an additional transition in the presence
of such substrates. CCR4 also displayed no difference in its activity
with capped or uncapped RNA substrates. These results indicate
that CCR4 recognition of its RNA substrates involves several features
of the RNA that could be sites in vivo for controlling the
rate of specific mRNA deadenylation.
*
This work was supported by National Institutes of Health
Grant GM41215 and HATCH Project H291. This is Scientific
Contribution 2155 from the New Hampshire Agriculture Experiment
Station.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.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of New Hampshire, Rudman Hall, Rm.
387, 46 College Rd., Durham, NH 03824. Tel.: 603-862-2427; Fax:
603-862-4013; E-mail: cldenis@cisunix.unh.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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