Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase.
Polyadenylation of messenger RNA precursors is an essential process in eukaryotes. Poly(A) polymerase (PAP), a member of the nucleotidyltransferase family that includes DNA polymerase beta, incorporates ATP at the 3' end of mRNAs in a template-independent manner. Although the structures of mammalian and yeast PAPs are known, their mechanism of ATP selection has remained elusive. In a recent bovine PAP structure complexed with an analog of ATP and Mn2+, strictly conserved residues interact selectively with the adenine base, but the nucleotide was found in a "non-productive" conformation. Here we report a second bovine crystal structure, obtained in the presence of Mg2+, where 3'-dATP adopts a "productive" conformation similar to that seen in yeast PAP or DNA polymerase beta. Mutational analysis and activity assays with ATP analogs suggest a role in catalysis for one of the two adenine-binding sites revealed by our structural data. The other site might function to prevent futile hydrolysis of ATP. In order to investigate the role of metals in catalysis we performed steady state kinetics experiments under distributive polymerization conditions. These tests suggest a sequential random mechanism in vitro in the presence of ATP and RNA, without preference for a particular order of binding of the two substrates. In vivo, however, where polyadenylation is processive and the primer does not dissociate from the enzyme, an ordered mechanism with the primer as the leading substrate is more likely.
Martin
Georges
G
Department of Cell Biology Biozentrum, University of Basel, Switzerland.
Möglich
Andreas
A
Keller
Walter
W
Doublié
Sylvie
S
D000225:Adenine / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D008958:Models, Molecular; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity
D011062:Polynucleotide Adenylyltransferase; D000225:Adenine