Thermodynamic Characterization of the Cooperativity of 40S Complex Formation during the Initiation of Eukaryotic Protein Synthesis

• Published in: Biochemistry (Easton) 1994-12, Vol.33 (50), p.15168-15177
• Main Authors: Parkhurst, Kay M, Hileman, Ronald E, Saha, Debabrata, Gupta, Naba K, Parkhurst, Lawrence J
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Binding Sites; Codon; Eukaryotic Initiation Factor-2 - metabolism; Fluorescence Polarization; Fluorescent Dyes; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate - metabolism; Molecular Sequence Data; Protein Biosynthesis; Proteins - metabolism; Ribosomes - metabolism; RNA, Messenger - metabolism; RNA, Transfer, Met - metabolism; Structure-Activity Relationship; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00254a028

The first step in mammalian protein synthesis is the formation of the 40S initiation complex, composed of the 40S ribosomal subunit (R), mRNA (M, here, a 10-mer oligoribonucleotide analogue containing the initiation codon), and the quaternary complex (Q, composed of eIF-2, GTP, Met-tRNA(fMet), and the ancillary protein factor Co-eIF-2C). The interdependence of the binding of R, M, and Q in forming the 40S complex is currently unclear. We have determined the thermodynamic parameters that characterize these interactions. The binary constants for R+M and Q+M were determined spectroscopically, measuring changes in the anisotropy of the fluorescence emission of 3'-fluorescein labeled M. The other binary constant, for Q+R, and the ternary constant were determined from Millipore filtration assays using radiolabeled Met-tRNA(fMet). The association constants for the binary reactions were as follows: Ka(Q,M) < or = 0.14 x 10(6) M-1, Ka(R,M) = 1.78 x 10(6) M-1, and Ka(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 [Ka(Q,R.M)/Ka(Q,R) > 62]. High cooperativity for this interaction occurs in either a single-site model or in lattice models for the binding of M to R. Data obtained 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 to 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 the complex.