Korneeva N L, Lamphear B J, Hennigan F L, Merrick W C, Rhoads R E
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport 71130-3932, USA.
J Biol Chem. 2001 Jan 26;276(4):2872-9. doi: 10.1074/jbc.M006345200. Epub 2000 Nov 1.
Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 x 10(5) m(-1) s(-1), 2.1 x 10(-3) s(-1), and 17 nm for the central site and 5.1 x 10(3) m(-1) s(-1), 1.7 x 10(-3) s(-1), and 330 nm for the COOH-terminal site.
真核生物翻译起始因子4G-1(eIF4G)在将mRNA募集到43S前起始复合物的过程中发挥关键作用。eIF4G有两个与RNA解旋酶eIF4A结合的位点,一个在中央结构域,一个在COOH末端结构域。分别包含这两个位点的重组eIF4G片段以1:1的化学计量比与eIF4A结合,但包含两个位点的片段以1:2的化学计量比与eIF4A结合。eIF3不干扰eIF4A与中央位点的结合。有趣的是,在相同浓度的游离eIF4A条件下,与包含两个eIF4A位点的eIF4G片段结合的eIF4A比与包含单个位点的片段结合的eIF4A总和还要多,这表明存在协同结合。eIF4A与包含COOH末端位点的eIF4G固定化片段的结合被包含中央位点的可溶性eIF4G片段竞争,这表明单个eIF4A分子不能同时与两个位点结合。通过表面等离子体共振测定每个位点的缔合速率常数、解离速率常数和解离平衡常数,发现中央位点分别为1.2×10⁵ m⁻¹ s⁻¹、2.1×10⁻³ s⁻¹和17 nm,COOH末端位点分别为5.1×10³ m⁻¹ s⁻¹、1.7×10⁻³ s⁻¹和330 nm。