In eukaryotic translation, initiation factors (eIFs) promote dissociation of vacant 80S (canonical initiation) or posttermination (recycling) ribosomes and assist binding of Met-tRNAiMet
and 5′-capped mRNA to the 40S subunit to form 43S and 48S preinitiation complexes (PICs), respectively (for reviews, see references 15
). eIF2 binding of Met-tRNAiMet
is dependent on GTP bound to its γ subunit, delivering it to the 40S subunit. The 43S complex additionally contains eIF1A, eIF1, eIF3, and eIF5, the latter three forming a multifactor complex (MFC) with the eIF2/GTP/Met-tRNAiMet
ternary complex (TC) (3
). Except for eIF3, which binds the solvent side of the 40S subunit (38
), these factors bind the decoding site of the 40S subunit and together play a role in positioning Met-tRNAiMet
in the P site. The cytoplasmic mRNA cap-binding complex eIF4F is made of three subunits, the major scaffold eIF4G, m7
G-cap-binding subunit eIF4E, and the mRNA helicase, eIF4A. eIF4F primes the 5′-proximal area of the m7
G-capped mRNA by the action of eIF4A and recruits the 43S complex to the primed region of the mRNA to form the 48S PIC. According to the scanning model, the 5′-proximal 48S complex migrates downstream along the mRNA, searching for the start codon.
Currently, much effort is devoted to understanding the mechanism by which these initiation factors coordinately regulate the state of the 48S PIC. During scanning, the PIC is presumed to be in the scanning-competent open state, and once a start codon is reached, it is shifted to the closed state, establishing tRNAiMet
:mRNA binding in the P site. Thus, the PIC in the closed state does not scan; instead, it tightly binds the selected start codon to promote formation of the 40S initiation complex (IC) (6
). eIF1 is present in the PIC in the open state and plays a major role in antagonizing stable tRNAiMet
:mRNA binding to the P site, thus suppressing transition to the closed state (27
). The release of eIF1 in response to AUG selection is considered to be the key regulatory event during the ribosome response to a start codon (10
). eIF5 promotes GTP hydrolysis of eIF2-GTP upon or subsequent to PIC formation (GTPase activation protein or GAP function), but the GDP and inorganic phosphate resulting from the hydrolysis remain bound to eIF2. Inorganic phosphate is released after eIF1 release, allowing eIF2-GDP to be ejected from the ribosome (2
While eIF1 release is considered primarily as a ribosomal response to start codon recognition, the contribution of other factors in retaining eIF1 in the open complex and facilitating its release on start codon selection remains elusive. Recently, a novel role for eIF5 in antagonizing eIF1 function by promoting its release has been proposed (27
), but the mechanism by which eIF5 promotes eIF1 release is unknown. Another open question is how mRNA binding to the PIC is stabilized with the mRNA entry channel open (30
eIF4G is the major scaffolding subunit of eIF4F, linking eIF4A to the m7
G-cap of the mRNA and thereby recruiting mRNA to the PIC (24
). Mammalian and most other metazoan eIF4G contain three HEAT domains, the first two of which serve as eIF4A-binding sites and the last as the binding site for Mnk eIF4E kinases (17
). Yeast eIF4G lacks the last two HEAT domains but retains the first HEAT domain, which is sufficient to bind eIF4A. Despite this structural difference, the functions of mammalian and yeast eIF4G are very similar, most notably in their ability to bind eIF4A, eIF4E, and the poly(A)-binding protein (Pab). In addition, conserved RS domains of eIF4G, here termed RS1 and RS2, are located N and C terminally to the MIF4G HEAT domain (26
). While the RS1 domain of mammalian eIF4G1 is shown to promote scanning for a start codon under the control of the internal ribosome entry site (31
), the precise role of these disordered segments is unknown.
The N-terminal tail (NTT) of eIF2β contains three lysine-rich segments called K-boxes (4
). eIF2β-NTT is the common binding site for eIF5 and eIF2Bε, promoting eIF2 TC recruitment to the ribosome (3
) and the reactivation of eIF2 by guanine nucleotide exchange (4
), respectively. Interestingly, eIF2β-NTT also binds mRNA via its K-boxes, suggesting a role for eIF2β in mRNA recruitment (20
). Moreover, depletion of eIF4G in yeast does not compromise mRNA binding to the 40S subunit in vivo
, suggesting that additional factors, including eIF2 and eIF3, are involved in stably anchoring mRNA to the 40S subunit (19
). Furthermore, alanine substitutions altering K-boxes increase the accuracy of start codon selection, suggesting that eIF2β is also involved in a subsequent step in AUG recognition (20
). The precise roles for eIF2β K-boxes in contributing to mRNA binding to the PIC and start codon selection have not been defined.
To answer the questions mentioned above, we have studied the positively charged, disordered segments of eIF4G and eIF2β, the RS1 domain of eIF4G, and the K-boxes of eIF2β, which we find bind eIF1, eIF5, and mRNA (7
). Our data indicate that eIF4G-RS1 interaction with eIF5 is directly responsible for eIF4G-mediated mRNA binding to the 43S complex, and that the eIF2β-K-boxes stabilize mRNA binding to the 40S subunit.
In selecting AUG start codons, we find that the mutation of eIF4G-RS1 or eIF5 increases the accuracy of the start codon. Our data support a model whereby the eIF2β-K-boxes and eIF4G-RS1, along with the eIF5 C-terminal domain (eIF5-CTD, or eIF5-C), are involved in shifting the ribosome to a closed state after mRNA binding. Based on previous studies and the data presented here, we propose a detailed protein-protein interaction model explaining how the rearrangement of interactions involving eIF2β-NTT and eIF4G-RS1 leads to eIF1 release in response to start codon recognition.