The objective of this research is to study the molecular mechanism of peptide chain initiation in animal cells and the roles of different factors in regulation of protein synthesis under different physiological conditions which include mitogen stimulation and viral infection. The first step in peptide chain initiation is the formation of a ternary complex between the eukaryotic peptide chain initiation factor 2 (eIF-2), Met-tRNAf and GTP; Met-tRNAf.eIF-2.GTP. The next step is the transfer of Met-tRNAf to the 40S ribosomal subunit and formation of Met-tRNAf.40S.mRNA complex. An important regulatory mechanism involves one or more eIF-2 kinases and an eIF-2 kinase inhibitor (a 67 kDa polypeptide;p67). eIF-2 kinases phosphorylate eIF-2 and thus inhibits protein synthesis and the increased availability of p67 renders eIF-2 resistant to eIF-2 kinase phosphorylation and thus promotes protein synthesis in the presence of eIF- 2 kinases. In this research, we propose to study: (I) The characteristics and factor requirements for ternary and Met-tRNAf.40S.mRNA complex formation. This work will be done in collaboration with Drs. John W.B. Hershey (Davis, CA) and William C. Merrick (Cleveland, OH). Using factor preparations and methodologies from three laboratories, we will attempt to arrive at a mutually agreeable definition for the characteristics and factor requirements for ternary and Met-tRNAf.40S.mRNA complex formation. (II) The roles and requirements of different factors for Met-tRNAf.40S complex formation with different mRNAs. Different natural and synthetic mRNAs will be used and attempts will be made to correlate the qualitative and quantitative requirements of different factors for Met-tRNAf.40S.mRNA complex formation with specific structural features such as secondary structure in the 5'-untranslated regions in mRNAs. (III) The mechanism of interaction of mRNAs with peptide chain initiation factors and 40S ribosomes. Attempts will be made to map the nucleotide sequences in different natural and synthetic mRNAs involved in binding to different protein factors and 40S ribosomes at different stages leading to Met- tRNAf.40S.mRNA complex formation. (IV) The roles of eIF-2 kinases and p67 in regulation of protein synthesis. The requirements of p67 in protein synthesis will be studied. Also, attempts will be made to determine qualitative and quantitative changes in p67 and eIF-2 kinases during mitogen stimulation and also during polio viral infection and relate these changes with the protein synthesis activities of the cells.
