Gene expression in eukaryotes is often controlled at the level of the initiation of translation. eIF3 plays a central role in this process. It is responsible for ribosome subunit dissociation, it stabilizes the initiator Met-tRNA on the small ribosomal subunit and it is required for mRNA binding to the ribosome. p33, an RNA-binding subunit of eIF3, has recently been cloned and partially characterized. p33 is part of the """"""""core"""""""" of the eIF3 complex and is an essential protein in yeast. The elucidation of the function of the p33 subunit, the function of eIF3, and the mechanism of translation initiation in yeast in a longer perspective will be sought in the research under this training period via these four specific aims: 1. Identify the physiological RNA target for p33 binding using SELEX. 2. Isolate high and low copy number extragenic suppressors of p33 mutants. 3. Use microarrays and kinetic RT-PCR to examine mRNA from polysome fractions in p33 mutants. . 4. Elucidate the structure/function of eIF3 by protein crosslinking and by forming partial complexes (e.g., p33, p39 and p93) and testing their activities in vitro.
