Human Eukaryotic Initiation Factor 2 (eIF2)-GTP-Met-tRNAi Ternary Complex and eIF3 Stabilize the 43S Preinitiation Complex [RNA]

September 22nd, 2014 by Sokabe, M., Fraser, C. S.

The formation of a stable 43S preinitiation complex (PIC) must occur to enable successful mRNA recruitment. However, the contributions of eIF1, eIF1A, eIF3 and the eIF2-GTP-Met-tRNAi ternary complex (TC) in stabilizing the 43S PIC are poorly defined. We have reconstituted the human 43S PIC and used fluorescence anisotropy to systematically measure the affinity of eIF1, eIF1A and eIF3j in the presence of different combinations of 43S PIC components. Our data reveal a complicated network of interactions that result in high affinity binding of all 43S PIC components with the 40S subunit. Human eIF1 and eIF1A bind cooperatively to the 40S subunit, revealing an evolutionarily conserved interaction. Negative cooperativity is observed between the binding of eIF3j and the binding of eIF1, eIF1A and TC with the 40S subunit. To overcome this, eIF3 dramatically increases the affinity of eIF1 and eIF3j for the 40S subunit. Recruitment of TC also increases the affinity of eIF1 for the 40S subunit, but this interaction has an important indirect role in increasing the affinity of eIF1A for the 40S subunit. Together, our data provide a more complete thermodynamic framework of the human 43S PIC and reveals important interactions between its components to maintain its stability.