In this study we show that residues 654–700 of b/PRT1 are sufficient for i/TIF34 binding and present, to our knowledge, the first atomic-resolution structure of the interaction between two essential core eIF3 subunits. Our structure reveals that the two major contacts between i/TIF34 and b/PRT1(654–700) occur on the bottom side of the i/TIF34 β-propeller through conserved residues in blades 5 and 6. Disrupting the first contact between D207 and D224 of i/TIF34 and the corresponding Y677 and R678 of b/PRT1 produces lethal or Ts−
phenotypes and severely diminishes MFC- and 40S-association of i/TIF34, and also that of g/TIF35, in vivo
(). Importantly, it also destabilizes binding of the rest of eIF3 and eIF5 to the 40S subunit (). Likewise, disrupting the second contact between W674 of b/PRT1 and the hydrophobic pocket of i/TIF34 confers essentially the same phenotypes (, Supplementary Figure S8D
, and data not shown). Together these results clearly indicate that both contacts between b/PRT1 and i/TIF34 critically stabilize association of the i/TIF34–g/TIF35 mini-module with the rest of eIF3, in close co-operation with those contacts that g/TIF35 makes with both subunits. This in turn ensures efficient loading of these two small eIF3 subunits onto the 40S where they are subsequently required for proper assembly of the 48S PICs.
Several important intermolecular bridges between yeast eIF3 and the solvent-exposed side of the 40S ribosome were previously identified, including those between the NTD of a/TIF32 and the small ribosomal protein RPS0A, the a/TIF32-CTD and helices 16–18 of 18S rRNA and RPS2 and RPS3, and the CTD of j/HCR1 and RPS2 (12
). Note that both RPS2 and 3 are situated near the mRNA entry pore (, upper panel). Besides them, the CTD of c/NIP1 and the b/PRT1-RRM were also shown to critically contribute to the eIF3 affinity for the 40S subunit; however, their binding partners remain to be identified (12
). Whether or not the remaining eIF3 subunits in i/TIF34 and g/TIF35 likewise participate in this functionally crucial eIF3-ribosome binding activity remained unclear until now. On the one hand a partial subcomplex composed of i/TIF34, g/TIF35 and b/PRT1 lacking its N-terminal RRM showed zero 40S-binding affinity in vivo
). On the other hand, another subcomplex comprising c/NIP1, the critical N-terminal half of a/TIF32, and eIF5 showed a substantial affinity for the 40S subunits in vivo
, though not as strong as that of the wt 6-subunit eIF3 (37
). Based on these findings and the data presented here, we propose that whereas the major and essential driving force of the 40S-binding affinity of yeast eIF3 lies in the three largest subunits, as proposed earlier (37
), i/TIF34 and g/TIF35 provide complementary 40S-binding activity that is required for stabilization of the entire 48S PICs. It is therefore conceivable that the proper establishment of all intermolecular bridges between eIF3 and the 40S ribosome is needed to ensure precise positioning of eIF3 on the small subunit and thereby flawless functioning of eIF3 not only in formation of 43S and 48S PICs, but also in the subsequent initiation steps.
Figure 9. A model of two eIF3 modules bound to the opposite termini of the scaffold b/PRT1 subunit situated near the mRNA entry channel of the 40S subunit. (Upper panel) The Cryo-EM reconstruction of the 40S subunit is shown from the solvent side with ribosomal (more ...)
This last notion resonates with our findings that the DD/KK
mutation produces severe Gcn−
phenotype owing to the robustly increased skipping of the AUG start codon (). It is assumed that this so-called leaky scanning phenotype results from an inability of the 48S PIC to switch from the open/scanning-conducive conformation of the 40S head region to the closed/scanning-arrested one that occurs upon AUG recognition and is strictly regulated by eIFs 1 and 1A (reviewed in (46
)). The conformational change upon scanning arrest is characterized by dissolution of the contact between RPS3 and helix 16 of 18S rRNA and reformation of the helix18–helix34-RPS3 connection designated as the latch on the mRNA entry channel (49
). Interestingly, the leaky scanning phenotype was also observed with mutations disrupting the web of interactions among the a/TIF32-CTD, the b/PRT1-RRM and j/HCR1 located most likely below the mRNA entry channel (12
) (, lower panel). Likewise, g/TIF35 was recently found to interact with RPS3 and RPS20, two subunits located above the mRNA entry channel (5
) (). This advocates that both termini of b/PRT1 and their associated eIF3 subunits can influence the precision and/or timing of this critical conformational transition upon AUG start codon recognition (see our model in , lower panel). We envisage three functional consequences of our mutants that could explain the leaky scanning as well as growth defects.
First, the lack of i/TIF34 and g/TIF35 may change the overall conformation and/or orientation of the rest eIF3 on the ribosome in a way that interferes with dynamics of the latch closing upon start codon recognition, allowing the 40S ribosome to skip the authentic AUG codon and continue scanning downstream. The aforementioned fact that g/TIF35 interacts with RPS20 and mainly with RPS3, which is one of the main components of the ‘latch mechanism’ (5
), is consistent with this hypothesis. If true, the scaffold b/PRT1 subunit would serve to connect two eIF3 modules at each of its termini as indicated in (a/TIF32-CTD–j/HCR1 at the N-terminal RRM, and i/TIF34–g/TIF35 at the C-terminal α-helix) that would work together and with c/NIP1 (39
) and other eIFs (46
) to fine-tune the AUG selection process.
The second contributor to the leaky scanning phenotype might originate from defects in the interaction of the PICs with mRNA in the background of the disrupted interactions between eIFs. It is evident now that eIF3 is crucial for productive mRNA recruitment (50
) and there is also evidence that eIF3 directly interacts with mRNA (5
). This might suggest that changes in how eIF3 interacts with the PICs could negatively affect the way the mRNA interacts with the mRNA binding channel having an ultimate impact on fidelity of start codon recognition.
The third contribution could arise from our finding that breaking the contact between b/PRT1 and i/TIF34 subunits leads to accumulation of erroneous PICs containing the TC and eIF1 (A), the amounts of which can be reduced by overexpressing TIF35
(C) or sui1G107R
(C). The fact that high dosage of g/TIF35 suppresses the Gcn−
and partially also the Slg−
phenotypes of tif34-DD/KK
but paradoxically does not better the assembly of the MFC and 48S PICs indicates that the presence of these aberrant complexes has a dominant negative effect on the initiation process reducing the overall fitness of mutant cells. Given the lack of any precedent for these repeatedly observable effects, we can only hypothesize that such forms of aberrant PICs would originate from simultaneous binding of all eIFs associated around eIF3 in the MFC to the ribosome followed by rapid dissociation of the unstably bound eIF3 (since lacking the i/TIF34–g/TIF35 mini-module) and eIF5 (eIF5 is known to bind to eIF3 very tightly (3
)). It is assumed that the MFC has to undergo a relatively extensive rearrangement when it associates with the 40S subunit (37
), as eIFs 1, 2, and most probably also 5 occur on the 40S interface side, whereas eIF3 binds to the solvent-exposed side [reviewed in (2
)]. Hence we can stipulate that eIF3 and eIF5 preferentially fall off during this rearrangement period. We further stipulate that the resulting TC-eIF1-rich PICs would have the TC locked in a conformation conducive to scanning but incompatible with initiation (the so-called the Pout
conformation of the TC), as recently described for some eIF1A mutants (53
), and as such they would be skipping the AUG start codons with dramatically increased frequency, as observed with our mutants. How to explain the paradox mentioned above? We propose that the increased dosage of g/TIF35 completely eliminates the MFC-driven PIC assembly pathway that is considered to be the most efficient way of the PIC formation (1
). As a consequence, PICs in the mutant cells overexpressing g/TIF35
must form solely by a less efficient yet still fully functional stochastic association of individual eIFs with the 40S ribosome, reminiscent of the bacterial initiation reaction, with no need for ribosomal rearrangement.
The fact that high dosage of sui1G107R
also reduced amounts of TC-containing PICs in tif34-DD/KK
(C) but, in contrast to high copy TIF35
, did not further destabilize the MFC in the mutant cells suggests a different molecular mechanism of suppression. In the light of earlier observations with reconstituted translational systems showing that mammalian eIF1 disrupts aberrantly formed PICs in vitro
), we could speculate that an increased dosage of eIF1 disrupts the aberrant PICs directly also in vivo
. In support, we found that the sui1G107R
mutant, which in contrast to other sui1
mutants decreases the fidelity of start codon recognition without increasing the rate of eIF1 release from the PICs, suppressed the Gcn−
and leaky scanning phenotypes of the DD/KK
mutant like wt eIF1 (A and B). These results imply that the eIF1 effect in DD/KK
cells requires its stable physical presence on the 40S ribosome, where it could directly manipulate the aberrant PICs. However, at odds with this scenario, overexpression of wt eIF1 had no impact on the amounts of the 40S-bound TC in tif34-DD/KK
. Hence at present we cannot offer any satisfactory explanation for this rather intriguing observation.
The aforementioned paradox that overexpressing TIF35 significantly suppresses the growth defect of the tif34-DD/KK mutant without rescuing the 40S association of eIF3 and eIF5 may also evoke a notion that the yeast cells can grow at wt rates with only a relatively small fraction (~40%) of the normal amounts of eIF3, eIF2 and eIF5 bound to 40S PICs. While we indeed cannot rule out this possibility, we think that in reality the 40S-binding of all factors in the living cells is most likely not as dramatically affected as we see in our cross-linking experiments, where the effect of a weaker 40S-association might get magnified by a mechanical breakage of cells and all other in vitro manipulations.
Finally, it should also be noted that we recently generated and analyzed two specific mutations in i/TIF34 and g/TIF35, neither of which had any effects on eIF3 and PIC assembly, that produced a severe slow scanning defect and significantly reduced processivity of scanning through stable secondary structures (5
). Neither of these scanning phenotypes was, however, found associated with the W674A
mutations analyzed here. This is not surprising, since the latter mutations affect translation by destabilizing the MFC and PIC formation, while the former mutations, which have no assembly defects, primarily disrupt eIF3 functions downstream of the 48S PIC assembly. This is consistent with the dual role of eIF3 as a central hub required for mRNA binding to the 43S PIC (48
) as well as a crucial player in scanning and AUG recognition (5
). It will now be intriguing to investigate the binding determinants of g/TIF35 in a partial subcomplex with i/TIF34 and b/PRT1-CTD and their interactions with the 40S subunit using the powerful combination of structural, genetic and biochemical approaches in order to continue replacing the hic sunt leones
on our illustrative model of eIF3 with real structures as presented in C.