Elucidating the Molecular Mechanics of Eukaryotic Translation Initiation and Its Control

点击翻译按钮获取中文摘要

The goal of our research group is to elucidate the molecular mechanisms underlying the initiation phase of protein synthesis in eukaryotic organisms. We use the yeast saccharomyces cerevisiae as a model system and employ a range of approaches - from genetics to biochemistry to structural biology - in collaboration with Alan Hinnebusch and Tom Devers labs at NICHD and several other research groups around the world. Eukaryotic translation initiation is a key control point in the regulation of gene expression. It begins when an initiator methionyl tRNA (Met-tRNAi) is loaded onto the small (40S) ribosomal subunit. Met-tRNAi binds to the 40S subunit as a ternary complex (TC) with the GTP-bound form of the initiation factor eIF2. Three other factors eIF1, eIF1A and eIF3 also bind to the 40S subunit and promote the loading of the TC. The resulting 43S pre-initiation complex (PIC) is then loaded onto the 5-end of an mRNA with the aid of eIF3 and the eIF4 group of factors the RNA helicase eIF4A; the 5-7-methylguanosine cap-binding protein eIF4E; the scaffolding protein eIF4G; and the 40S subunit- and RNA-binding protein eIF4B. Both eIF4A and eIF4E bind to eIF4G and form the eIF4F complex. Once loaded onto the mRNA, the 43S PIC is thought to scan along the mRNA in search of an AUG start codon. This process is ATP-dependent and likely requires multiple RNA helicases, including the DEAD-box protein Ded1p. Recognition of the start site begins with base pairing between the anticodon of tRNAi and the AUG codon. This base pairing then triggers downstream events that commit the PIC to continuing initiation from that point on the mRNA. These events include ejection of eIF1 from its binding site on the 40S subunit, movement of the C-terminal tail (CTT) of eIF1A, and release of phosphate from eIF2, which converts it to its GDP-bound state. In addition, the initiator tRNA moves from a position that is not fully engaged in the ribosomal P site (termed P(OUT)) to one that is (P(IN)) and the PIC as a whole converts from an open conformation that is conducive for scanning to a closed one that is not. At this stage eIF2GDP dissociates from the PIC and eIF1A and a second GTPase factor, eIF5B, coordinate joining of the large ribosomal subunit to form the 80S initiation complex. eIF5B hydrolyzes GTP, which appears to result in a conformational reorganization of the complex, and then dissociates along with eIF1A. This year we made significant progress on studies of the N-terminal tail (NTT) or eIF1. This tail appears to play a critical role in responding to recognition of the start codon in the mRNA by the PIC. We used an alanine-scanning mutagenesis approach to determine the effect of changing each amino acid in the tail to alanine. We have identified a variety of phenotypes of these mutants, from lethality to effects on the fidelity of start codon recognition. We are now in the process of studying the effects of key NTT mutations on the function of eIF1 in the in vitro reconstituted yeast translation initiation system in order to help elucidate the mechanistic roles of individual side chains in the NTT. We have also made progress on our in vitro RecSeq system for measuring the efficiency of mRNA recruitment genome wide. This system allows us to isolate the mRNA recruitment steps of translation initiation from later steps in the process and events such as mRNA synthesis or decay, and to actually measure the rates of recruitment of each mRNA simultaneously. Because the system is reconstituted, we can change the concentrations of components or replace them with mutant versions in order to conduct detailed mechanistic analyses.