Structural and enzymatic characterization of the yeast mRNA decapping enzyme, Dcp2.

摘要

mRNA turnover represents a fundamental point of post-transcriptional control of gene expression in eukaryotes. Decapping is a highly regulated, irreversible step in mRNA decay and is involved in many different decay pathways. The yeast mRNA decapping enzyme is comprised of the catalytic subunit, Dcp2, in complex with Dcp1, an obligate in vivo activator. The goal of this study is to characterize the mechanism of mRNA decapping by Dcp2 and the role of activators.;We embarked on this study by establishing an in vitro model system for decapping. Reconstituting this system required purifying the decapping holoenzyme and the catalytic subunit, preparing radiolabeled RNA substrate and determining optimal assay conditions. This assay allowed us to investigate how Dcp2 binds and recognizes its mRNA substrate, as well as the role of Dcp1 as an activator of decapping, by examining the various kinetic and thermodynamic parameters of this process. We discovered that Dcp2 contains an extensive, highly charged RNA binding surface extending from the active site through a channel to the dorsal side of the protein. This surface is bipartite in function: the dorsal surface binds the RNA body and provides the majority of the binding energy, whereas the cap proximal nucleotides are involved in the catalytic mechanism.;We also determined that Dcp1 stimulates decapping during the catalytic step, wherein Dcp2 undergoes an open-to-closed transition into a catalytically active conformation. This is supported by our result that the cap is recognized during the transition state, suggesting closure promotes cap recognition and enzyme activity. This represents a possible fundamental point of control of this process as activators could shift the balance by promoting the closed, active form of the enzyme. We began studies on one such activator of decapping, Edc3, and discovered that it directly interacts with Dcp2 to activate decapping, though further studies are necessary for determine which step in the catalytic cycle is affected.;This study is the first to mechanistically examine the decapping enzyme, Dcp2, using a multidisciplinary approach involving enzymology, biochemistry, biophysics, and genetics. Our goals were to investigate the structural and biochemical basis for RNA recognition and the mechanism of activation. These studies provide the first insights into control of decapping at the molecular level.