Template-independent elongation of the 3' end of mRNA by poly(A) polymerase (Pap) occurs in virtually all organisms. In eukaryotes, the non-coding mRNA extensions added by Pap serve as molecular handles, which interact with nuclear export, translation and mRNA degradation machinery, and strongly effect mRNA stability and translational efficiency. Our recent crystal structure of yeast poly(A) polymerase (Pap1) in complex with the non-extendable ATP analog 3-dATP revealed the catalytic mechanism for base addition, but not the basis for adenosine specificity, the path of the poly(A) tail through the large cleft surrounding the active site, or the structural basis for the high degree of processivity exhibited in vitro by purified Pap1. Pap1 is an excellent model for understanding processivity, since unlike most other polymerases, including mammalian Pap, it does not require additional proteins to achieve processivity. We propose to use x-ray crystallography as well as fluorescence resonance energy transfer, cross-linking, and activity measurements to study the relationship between the structure of Pap1 and its ability to specifically and processively elongate mRNA. We also propose work aimed at a structural understanding of how Pap1 is regulated by two other components, Fip1 and Yth1, of the mRNA cleavage/polyadenylation complex. Our preliminary crystallographic results suggest a basis for nucleotide specificity. Also, we have very recently identified a unexpected RNase activity in highly purified Pap1. Polyadenylation is a major regulator of protein expression. The work we propose will provide a dynamic model of the molecular events associated with this important regulatory process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065972-06
Application #
7088811
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Ikeda, Richard A
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
6
Fiscal Year
2006
Total Cost
$270,857
Indirect Cost
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
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