Division of Biology
Kansas State University
Molecular basis of control of translational initiation in eukaryotes (2012-2013)
Cancer cells acquire a high rate of protein synthesis due to mutations altering the activity or expression of eukaryotic translation initiation factors (eIF). This study aims to identify the “bottleneck” of initiation function. If we can solve the structure of the eIF complex in Aim 2, in silico screening will list a handful of chemicals that sterically inhibit the critical interaction in the complex, a significant step towards drug discovery.
The ribosome pre-initiation complex (PIC) conformational change in response to start codon selection is a recently discovered key event determining the rate and fidelity of translation initiation. eIF1 (~10 kDa) is the critical factor in this regulation, preventing the shift from the open, scanning-competent state to the closed, scanning-incompetent state. Once the start codon is recognized, eIF2ß-N-terminal tail (NTT) binds eIF5-CTD, severing eIF1 from the local assembly, and thereby alleviating the antagonistic function of eIF1 against the shift to the closed state. The central hypothesis of this grant is that the rearrangement of interactions involving eIF1, eIF5-CTD, and the disordered segments, eIF3c-NTT and eIF2ß-NTT, regulate start-codon induced changes from the open to closed state. We previously identified eIF5-binding face of eIF1 termed the KH area by NMR chemical shift mapping. By a similar approach, we now identify a distinct eIF3c-binding face of eIF1. We also localize distinct eIF5- and eIF1-binding sites within eIF3c-NTT. Based on these and other data, we have configured detailed interaction models explaining how the rearrangement of protein-protein interactions unmasks the ribosome-binding face of eIF1 at the initial assembly stage, and antagonizes the ribosome-binding in response to AUG selection, promoting eIF1 release from the ribosome. We will test these models in this grant. The specific aims are:
- To characterize the newly identified eIF3c-binding face of eIF1 by genetics and biochemistry
- To study the key regulatory interactions involving eIF3c-NTT by in vitro reconstitution
Aim 2 involves NMR spectroscopy and X-ray crystallography to be performed at the COBRE PSF facilities.