Regulation of GTP hydrolysis prior to ribosomal AUG selection during eukaryotic translation initiation

Abstract

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S . Met-tRNA i . eIF2 . GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S . eIF1 . eIF1A . eIF3 . Met-tRNA i . eIF2 . GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5 0 -cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.

Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action

“…eIF-2 is composed of three subunits (␣, ␤, and ␥); it binds the Met-tRNAi in a GTP-dependent manner to form the ternary complex, which joins the 40S subunit (153,236). Once Met-tRNAi is delivered, eIF-2 is released from the 48S initiation complex after eIF-5-promoted GTP hydrolysis (153,236). Inactive eIF-2-GDP complexes are continuously regenerated by GDP-to-GTP exchange in a process catalyzed by the GTP exchange factor eIF-2B.…”

Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2′ 5′-oligoadenylate synthetase family

“…The kinase domain is connected to the dsRBMs by an 80 residue flexible linker, thought to enable PKR dimerization [160]. PKR dimerization enables trans auto--phosphorylation of the kinase domain of inactive monomers, a requirement for enzymatic activation [161]. The Ser/Thr kinase needs phosphorylation at both Thr446 and Thr451 but other phosphorylation sites (for example, Tyr residues at 101, 162 and 293) also regulate enzymatic activity [162--164].…”

Translation factor control of ribosome conformation during start codon selection

“…The reconstituted system used does not include eIF3, so eIF3-dependent processes that would be important in vivo cannot be assayed. The system also lacks eIF4F, which has a critical role in loading the 43S complex onto mRNA in vivo, and may have additional roles in regulating TC GTPase activity (He et al 2003;Majumdar and Maitra 2005).…”

“…The exchange of GDP bound to eIF2 for GTP, with release of eIF5, by eIF2B is also indicated, as is the reformation of TC by binding of eIF2•GTP to Met-tRNA i Met . selection, the eIF2-mediated hydrolysis of GTP in the TC that must occur is assisted by eIF5, which acts as a GTPase activator protein (GAP) (Das et al 2001;Paulin et al 2001;Majumdar and Maitra 2005), and eIF1 appears to negatively affect this interaction (see below). But surprisingly, start codon selection is not strictly coupled to the hydrolysis event; it appears that hydrolysis can occur in the PIC during scanning, but the P i formed is not released and instead remains associated, so that there is equilibrium between GTP and GDP-Pi associated with TC ( Fig.…”