Coactivator regulation of active site chemistry in the mRNA decapping enzyme Dcp2
- Author(s): Aglietti, Robin
- Advisor(s): Gross, John D
- et al.
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.