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Molecular and Cellular Biology, October 1998, p. 5942-5951, Vol. 18, No. 10
Department of Molecular Biology and
Microbiology1 and
Department of
Biochemistry,2 Tufts University School of
Medicine, Boston, Massachusetts 02111-1800
Received 9 June 1998/Accepted 10 July 1998
The interaction of the Fip1 subunit of polyadenylation factor I
with the Saccharomyces cerevisiae poly(A) polymerase (PAP) was assayed in vivo by two-hybrid analysis and was found to involve two
separate regions on PAP, located at opposite ends of the protein sequence. In vitro, Fip1 blocks access of the RNA primer to an RNA
binding site (RBS) that overlaps the Fip1 carboxy-terminal interaction
region and, in doing so, shifts PAP to a distributive mode of action.
Partial truncation of this RBS has the same effect, indicating that
this site is required for processivity. A comparison of the utilization
of ribo- and deoxyribonucleotides as substrates indicates the existence
on PAP of a second RBS which recognizes the last three nucleotides at
the 3' end of the primer. This site discriminates against
deoxyribonucleotides at the 3' end, and interactions at this site are
not affected by Fip1. Further analysis revealed that the specificity of
PAP for adenosine is not simply a function of the ATP binding site but
also reflects interactions with bases at the 3' end of the primer and
at another contact site 14 nucleotides upstream of the 3' end. These
results suggest that the unique specificity of PAP for ribose and base,
and thus the extent and type of activity with different substrates,
depends on interactions at multiple nucleotide binding sites.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Processivity of the Saccharomyces
cerevisiae Poly(A) Polymerase Requires Interactions at the
Carboxyl-Terminal RNA Binding Domain
*
Corresponding author. Mailing address: Department of
Molecular Biology and Microbiology, Tufts University School of
Medicine, 136 Harrison Ave., Boston, MA 02111-1800. Phone: (617)
636-6935. Fax: (617) 636-0337. E-mail:
cmoore{at}opal.tufts.edu.
Molecular and Cellular Biology, October 1998, p. 5942-5951, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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