Mutations in the middle domain of yeast poly(A) polymerase affect interactions with RNA but not ATP.
The eukaryotic poly(A) polymerase (PAP) is responsible for the posttranscriptional extension of mRNA 3' ends by the addition of a poly(A) tract. The recently published three-dimensional structures of yeast and bovine PAPs have made a more directed biochemical analysis of this enzyme possible. Based on these structures, the middle domain of PAP was predicted to interact with ATP. However, in this study, we show that mutations of conserved residues in this domain of yeast PAP, Pap1, do not affect interaction with ATP, but instead disrupt the interaction with RNA and affect the enzyme's ability to process substrate lacking 2' hydroxyls at the 3' end. These results are most consistent with a model in which the middle domain of PAP interacts directly with the recently extended RNA and pyrophosphate byproduct.
Zhelkovsky
Alexander
A
Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences, Boston, Massachusetts 02111, USA.
Helmling
Steffen
S
Bohm
Andrew
A
Moore
Claire
C
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D009154:Mutation; D000073718:Pancreatitis-Associated Proteins; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics*
D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012313:RNA; D000255:Adenosine Triphosphate; D011062:Polynucleotide Adenylyltransferase