Biophysical studies of the translation initiation pathway using immobilised mRNA analogues

A growing number of biophysical techniques use immobilized reactants for the quantitative study of macromolecular reactions. Examples of such approaches include surface plasmon resonance, atomic force microscopy, total reflection fluorescence microscopy, and others. Some of these methods have already been adapted for work with immobilized RNAs, thus making them available for the study of many reactions relevant to translation. Published examples include the study of kinetic parameters of protein/RNA interactions and the effect of helicases on RNA secondary structure. The common denominator of all of these techniques is the necessity to immobilize RNA molecules in a functional state on solid supports. In this chapter, we describe a number of approaches by which such immobilization can be achieved, followed by two specific examples for applications that use immobilized RNAs

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA: EFFECTS OF eIF4B AND POLY(A)-BINDING PROTEIN*

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (kon) and dissociation rate constant (koff) for eIF4F binding to IRES were 59 ± 2.1 μm−1 s−1 and 12.9 ± 0.3 s−1, respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F ∼3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES

The mRNA cap-binding protein eIF4E in post-transcriptional gene expression

Eukaryotic initiation factor 4E (eIF4E) has central roles in the control of several aspects of post-transcriptional gene expression and thereby affects developmental processes. It is also implicated in human diseases. This review explores the relationship between structural, biochemical and biophysical aspects of eIF4E and its function in vivo, including both long-established roles in translation and newly emerging ones in nuclear export and mRNA decay pathways

Kinetic Mechanism for Assembly of the m7GpppG·eIF4E·eIF4G Complex*S⃞

Interaction of the mRNA cap with the translational machinery is a critical and early step in the initiation of protein synthesis. To better understand this process, we determined kinetic constants for the interaction of m7GpppG with human eIF4E by stopped-flow fluorescence quenching in the presence of a 90-amino acid fragment of human eIF4G that contains the eIF4E-binding domain (eIF4G(557–646)). The values obtained, kon = 179 × 106 m–1 s–1 and koff = 79 s–1, were the same as reported previously in the absence of an eIF4G-derived peptide. We also used surface plasmon resonance to determine kinetic constants for the binding of eIF4E to eIF4G(557–646), both in the presence and absence of m7GpppG. The results indicated that eIF4G(557–646) binds eIF4E and eIF4E·m7GpppG at the same rate, with kon = 3 × 106 m–1 s–1 and koff = 0.01 s–1. Our data represent the first full kinetic description of the interaction of eIF4E with its two specific ligands. The results demonstrate that the formation of the m7GpppG·eIF4E·eIF4G(557–646) complex obeys a sequential, random kinetic mechanism and that there is no preferential pathway for its formation. Thus, even though eIF4G(557–646) binds eIF4E tightly, it does not increase the affinity of eIF4E for m7GpppG, as has been claimed in several previous publications. We did, in fact, observe increased binding to m7GTP-Sepharose in the presence of eIF4G(557–646), but only with recombinant eIF4E that was prepared from inclusion bodies

Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands

The activity of the eukaryotic translation initiation factor eIF4E is modulated through conformational response to its ligands. For example, eIF4G and eIF4E-binding proteins (4E-BPs) modulate cap affinity, and thus physiological activity of eIF4E, by binding a site distal to the 7-methylguanosine cap-binding site. Further, cap binding substantially modulates eIF4E's affinity for eIF4G and the 4E-BPs. To date, only cap-bound eIF4E structures were reported. In the absence of structural information on the apo form, the molecular underpinnings of this conformational response mechanism cannot be established. We report here the first cap-free eIF4E structure. Apo-eIF4E exhibits structural differences in the cap-binding site and dorsal surface relative to cap-eIF4E. Analysis of structure and dynamics of apo-eIF4E, and changes observed upon ligand binding, reveal a molecular basis for eIF4E's conformational response to these ligands. In particular, alterations in the S4-H4 loop, distal to either the cap or eIF4G binding sites, appear key to modulating these effects. Mutation in this loop mimics these effects. Overall, our studies have important implications for the regulation of eIF4E