THERMODYNAMIC CHARACTERIZATION OF THE COOPERATIVITY OF 40S COMPLEX-FORMATION DURING THE INITIATION OF EUKARYOTIC PROTEIN-SYNTHESIS

The first step in mammalian protein synthesis is the formation of the 40S initiation complex, composed of the 40S ribosomal subunit (R), mRN A (M, here, a 10-mer oligoribonucleotide analogue containing the initi ation codon), and the quaternary complex (Q, composed of eLF-2, GTP, M et-tRNA(f)(Met), and the ancillary protein factor Co-eIF-2C). The inte rdependence of the binding of R, M, and Q in forming the 40S complex i s currently unclear. We have determined the thermodynamic parameters t hat characterize these interactions. The binary constants for R + M an d Q + M were determined spectroscopically, measuring changes in the an isotropy of the fluorescence emission of 3'-fluorescein labeled M. The other binary constant, for Q + R, and the ternary constant were deter mined from Millipore filtration assays using radiolabeled Met-tRNA(f)( Met). The association constants for the binary reactions were as follo ws: K-a(Q,M) less than or equal to 0.14 x 10(6) M(-1), K-a(R,M) = 1.78 x 10(6) M(-1), and K-a(Q,R) = 0.94 x 10(6) M(-1). The binding of Q to R.M was markedly greater than that of Q to R [K-a(Q,R.M)/K-a(Q,R) > 6 2]. High cooperativity for this interaction occurs in either a single- site model or in lattice models for the binding of M to R. Data obtain ed using five other RNA 10-mers, each with the sequence altered at the AUG codon, suggest that this cooperativity is AUG dependent. The data are consistent with a scheme in which mRNA and Q bind independently t o the 40S ribosome, but when the AUG codon is properly aligned with Q, a conformational change results in a 2.4 kcal/mol stabilization of th e complex.