Coactivator regulation of active site chemistry in the mRNA decapping enzyme Dcp2

Regulation of mRNA half-life is a crucial control point of gene expression. Removal of the protective 5' methylguanosine cap is a committed step in the 5'-3' decay pathway, which is carried out by the decapping enzyme Dpc2. Although Dcp2 is sufficient for decapping in vitro, its activity is greatly increased by coactivators; here the activation of Dcp2 is explored through several means. A thorough study of the chemistry of decapping is presented: multiple crystal structures implicate specific active site residues of Dcp2 in catalysis metal binding and enzyme kinetics identify key residues involved in the acid/base chemistry of decapping. Further, a metal binding loop is implicated in conformational changes coupled to Dcp2's catalytic cycle using pH-dependent NMR spectroscopy and molecular dynamics simulations. In addition we hypothesized and tested direct effects of other potential regulators of decapping by in vitro assay and binding studies.;In addition, two potential decapping activation pathways are explored. First, a hypothesized interaction between the decapping complex and nonsense-mediated mRNA decay factor Upf1 is explored through GST pull-downs, and enzyme kinetics. Additionally, some evidence suggests the possibility that an extended ribonucleoprotein complex containing mRNA decay factors Pat1/Lsm1-7 and Xrn1 may directly affect the activity of Dcp2; this hypothesis is tested using an in vitro decapping assay. However, future experiments are needed to fully characterize coactivator regulation of Dcp2 within the context of the cell.