Featured Article - April 2009
Short description: A combination of NMR, mutagenesis and biochemistry reveals the dynamic nature of the eIF4A/4G/4H helicase complex.Cell 136, 447-470 (2009)
Protein synthesis is tightly controlled, and one of the main steps at which it is regulated is the initiation of mRNA translation. Initiation is usually the rate-limiting step in translation; it is the stage at which the preinitiation complex containing the small (40S) ribosomal subunit assembles and is recruited to the 5′ cap of mRNA. Once recruited, the complex scans along the mRNA in the 3′ direction in search of the start codon.
Scanning is hampered by secondary structure within the 5′ untranslated region of mRNA, and an ATP-dependent helicase activity is needed to facilitate the binding of the preinitiation complex and improve scanning. The helicase is provided by the eukaryotic initiation factor eIF4A. This has very low helicase activity on its own but is much more efficient when complexed with the accessory proteins eIF4B, eIF4E, eIF4G and eIF4H. eIF4A consists of two helicase domains, both of which bind RNA and ATP.
Despite the general functions of eIF4A, eIF4G and eIF4E having been known for decades, and their individual structures having been solved, the structure of the multiprotein complex has remained elusive. This is probably because contacts within the complex are constantly rearranged as it moves along and unwinds mRNA, which makes crystallization difficult.
Marintchev et al. now reveal the topology of the human eIF4A/4G/4H helicase complex through a combination of NMR, site-directed mutagenesis and biochemical assays. They show that it comprises a dynamic network of multiple weak but specific interactions that are continuously rearranged during the ATP-binding and hydrolysis cycle of the helicase.
They also demonstrate that the stable association of eIF4H with eIF4A and eIF4G requires the presence of ATP. It was already known that the entire complex cycles through three distinct states: ATP-bound, ADP-bound and nucleotide free, but this study indicates that the overall domain orientation remains roughly similar in all three states.
Marintchev et al. probed various interactions between the subunits to show that the accessory proteins modulate the affinity of eIF4A for ATP by interacting with both of its helicase domains and by promoting either the closed, ATP-bound, conformation or the open, nucleotide-free, conformation. They also found that helicase tethers eIF4A to mRNA, and that binding of eIF4H to single-stranded mRNA behind eIF4A prevents mRNA annealing and promotes the unidirectional translocation of eIF4A.
The most striking finding is that the complex contacts the mRNA on both sides of the nucleotides located in the ribosome's decoding sites, and the authors propose that different subunits are located in front of and behind the 40S subunit during scanning. Such a conformation could be achieved by wrapping the mRNA around the 'neck' of the 40S subunit, bringing both ends of the mRNA close together. This would position the complex at the mRNA entry channel in the appropriate location for unwinding RNA secondary structure.
A. Marintchev et al. Topology and regulation of the human eIF4A/4G/4H helicase complex in translation initiation.
Cell 136, 447-470 (2009).