title: Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Nebraska--Lincoln, 68588-0304.
authors.forename: K M
authors.initials: KM
authors.lastname: Parkhurst
authors.forename: R E
authors.initials: RE
authors.lastname: Hileman
authors.forename: D
authors.initials: D
authors.lastname: Saha
authors.forename: N K
authors.initials: NK
authors.lastname: Gupta
authors.forename: L J
authors.initials: LJ
authors.lastname: Parkhurst
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D003062:Codon; D015852:Eukaryotic Initiation Factor-2; D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D006160:Guanosine Triphosphate