1Q78

Crystal structure of poly(A) polymerase in complex with 3'-dATP and magnesium chloride

Summary for 1Q78

• Entry DOI: 10.2210/pdb1q78/pdb
• Related: 1Q79
• Descriptor: Poly(A) polymerase alpha, MAGNESIUM ION, 3'-DEOXYADENOSINE-5'-TRIPHOSPHATE, ... (4 entities in total)
• Functional Keywords: mrna processing, nucleotidyl transferase, transferase
• Biological source: Bos taurus (cattle)
• Cellular location: Nucleus: P25500
• Total number of polymer chains: 1
• Total formula weight: 59730.79

Authors

Martin, G.,Moglich, A.,Keller, W.,Doublie, S. (deposition date: 2003-08-16, release date: 2004-09-07, Last modification date: 2024-11-13)

Primary citation

Martin, G.,Moglich, A.,Keller, W.,Doublie, S.
Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase.
J.Mol.Biol., 341:911-925, 2004

PubMed Abstract: 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.

PubMed: 15328606
DOI: 10.1016/j.jmb.2004.06.047

Experimental method

X-RAY DIFFRACTION (2.8 Å)