Article

Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis.

Department of Biochemistry, Tufts School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
Structure (impact factor: 6.35). 10/2007; 15(9):1117-31. DOI:10.1016/j.str.2007.07.010 pp.1117-31
Source: PubMed

ABSTRACT We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.

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Keywords

aspartic acid 154
 
catalytic strategies utilized
 
catalytically
 
closed conformation
 
conformational change
 
correct nucleotide
 
domain closure
 
enzyme state
 
extensive interactions
 
five residue poly(A)
 
MgATP
 
nucleotidyltransfer reaction utilizing CTP
 
pyrophosphorolysis
 
significant domain movement
 
three polymerase domains
 
yeast poly(A)
 

Paul B Balbo