Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis

Kay M. Parkhurst, Ronald E. Hileman, Debabrata Saha, Naba K. Gupta, Lawrence J. Parkhurst

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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-tRNAf Met, 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-tRNAf Met. The association constants for the binary reactions were as follows: Ka(Q,M) ≤ 0.14 × 106 M-1, Ka(R,M) = 1.78 × 106 M-1, and Ka(Q,R) = 0.94 × 106 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.

Original languageEnglish (US)
Pages (from-to)15168-15177
Number of pages10
JournalBiochemistry
Volume33
Issue number50
StatePublished - 1994

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Thermodynamics
Codon
Eukaryotic Small Ribosome Subunits
Oligoribonucleotides
Messenger RNA
Fluorescence Polarization
Initiator Codon
Guanosine Triphosphate
Fluorescein
Ribosomes
Assays
Proteins
Anisotropy
Stabilization
Fluorescence
Association reactions
RNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis. / Parkhurst, Kay M.; Hileman, Ronald E.; Saha, Debabrata; Gupta, Naba K.; Parkhurst, Lawrence J.

In: Biochemistry, Vol. 33, No. 50, 1994, p. 15168-15177.

Research output: Contribution to journalArticle

Parkhurst, Kay M. ; Hileman, Ronald E. ; Saha, Debabrata ; Gupta, Naba K. ; Parkhurst, Lawrence J. / Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis. In: Biochemistry. 1994 ; Vol. 33, No. 50. pp. 15168-15177.
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