Preliminary kinetic insights into the formation of eIF3 and its binding to the 40S

Figure 5.1. Schematic model of yeast MFC. In this mode, Tif35 is repositioned in a way that binds to  Prt1 via Tif34 (compared with Figure 1.7). Numbers and black lines represent the boundaries of the  proteolytic fragments. 

5.3. Preliminary kinetic insights into the formation of eIF3 and its binding to the 40S subunit using in vitro reconstituted fluorescence-labeled eIF3

Despite detailed kinetic studies of translation initiation events in prokaryotes, eukaryotic initiation is a challenge owing to its higher complexity. Contrary to three prokaryotic factors (IFs 1-3) there are around ten eukaryotic initiation factors, some of which encompassing more than one subunit, which finely regulate this process. To reduce the complexity, a short unstructured RNA is used which bypasses the requirement for some eIFs. eIF3, as the largest initiation factor, plays a crucial role in different aspects of initiation. However, due to its complexity and problems tied with its purification (such as co-purification of the GAP eIF5), eIF3 has not been subjected to many studies. Having in hand the in vitro reconstituted recombinant eIF3, the kinetics of initiation complex formation could be dissected using fluorescence-labeling of the largest subunit of eIF3, Tif32.

  Figure 5.2. Sequence based alignment of eIF3a from six different species. The following organisms have  been  used:  Homosapiens  sapiens  (HUMAN),  Mus  musculus  (MOUSE),  Xenopus  laevis  (XENLA),  Drosophila  melanogaster  (DROME),  Schizosaccharomyces  cerevisiae  (SCHPO)  and  Saccharomyces  cerevisiae (YEAST). Note the conservation of the N‐terminal region among different species.  

The first kinetic insights into the formation of the eIF3 complex were obtained between two large subunits of eIF3, Nip1 and Tif32 (Figure 2.5 A). The resulting curve could be fitted to a double exponential function with a rapid first phase followed by a moderate second phase lasting almost the whole experiment with apparent binding constant of 1.832 s^-1. To obtain a more complete picture of the binding, the dissociation constant (Kd) and off rate (koff) have to be measured using ITC and chase experiments, respectively. Stepwise formation of the complex and comparison of the resulting on and off rates will provide the keys to elucidate the mode of complex formation.

  Figure 5.3. Sequence based alignment of eIF3c from six different species. The organisms used for  alignment are the same as Figure 5.2. Note the conservation of the C‐terminal region among different  species.  

Tif32, Prt1 and Nip1 are shown to form the core of eIF3 required for its association with the ribosome in a manner stimulated by Hcr1 (Valásek et al. 2003; Valásek et al. 2001). Formation of this complex with the labeled Tif32 in the absence or presence of 40S subunit, followed by a chase of unlabeled Tif32 suggested an effect of the ribosome on the complex (Figure 2.5 B). The observed decrease in the fluorescence in the presence of 40S subunit and not in its absence might indicate structural rearrangements of eIF3 bound to the ribosome compared to free eIF3 which facilitates dissociation and reassociation of Tif32 from the complex. However, using this data we cannot rule out the possibility that in the presence of 40S subunit the positioning of the fluorophore is different leading to the observed difference between free and in-complex Tif32.

5.4. Preliminary EM studies on eIF3

In order to gain structural insights into the pentameric eIF3 complex either alone or in 40S subunit bound state, the most prominent approach is electron microscopy. Human eIF3, a complex of 800 kDa, was studied by electron microscopy at low resolution (Siridechadilok et al.

2005). However, as the ribosome-bound complex could not resist the grid-preparation treatments, it was model it on the ribosome. Yeast eIF3 is less than half of its human homolog in size, which makes it a difficult sample for EM. Our reconstituted eIF3 also dissociated into

several sub-particles in initial studies yielding an inhomogeneous sample (Figure 2.6 A).

However, applying the GraFix method (Kastner et al. 2005) helped to stabilize the complex resulting in visually much better samples with more defined particles (Figure 2.6 B). Preliminary 3D reconstruction has obtained a particle with two monomers which are related to each other by a two-fold symmetry (Figure 5.5 A-B).

To structurally compare recombinant and native eIF3s (eIF3^rec vs. eIF3^nat, respectively), they were analyzed by EM. Initial classification of the 2D projections has yielded class-averages which in many cases resemble a five-lobed particle in the case of eIF3^nat (Figure 5.4). The longest dimension of these particles is about 200 Å, very similar to that of eIF3^rec (Figure 2.9).

Due to the complexity and flexibility of the sample, the advantage of the structural rigidity of the ribosome was exploited. For this, eIF3^nat-40S complex was prepared and used for cryo-EM studies. These images will be used for reconstruction of eIF3^nat-40S, which in turn would help to solve the structure of free eIF3^nat. The latter structure could be then used to reconstruct eIF3^rec structure and its subcomplexes more reliably. Comparing the structure of eIF3^rec and eIF3^nat-40S will provide insight to the binding site of different subunits of eIF3 on the 40S subunit and shed light on eukaryotic translation initiation process.

  Figure 5.4. 2D analysis of the natively purified eIF3. Multiple class averages of cryogenic samples of  eIF3^nat show apparently 5‐lobed particles. 

By initial comparison of the 2D class averages of eIF3^nat and 3D reconstruction of eIF3^rec, it appears that 2D projections of eIF3^nat are akin to the back-projections of a monomer of eIF3^rec reconstruction. This suggests the possibility of using the monomer of eIF3^rec as the initial model for reconstruction of free or 40S-bound eIF3^nat. Preliminary inspections of the 40S-eIF3^rec 3D reconstruction revealed an extra density at the solvent-exposed side of the 40S between the head

and the body which penetrates into the inter-subunit surface (Figure 5.5 C-E). Docking of the density of the model of yeast 40S subuinit (as a part of an 80S elongating ribosome; PDB code 1S1H; Spahn et al. 2004) into the map of eIF3^nat-40S also suggests that this density most probably does not belong to the 40S subunit. The density of eIF3^rec monomer covers some parts of this extra density on eIF3^nat-40S, so that it can be at least partially assigned to eIF3. Further data processing, both on eIF3^rec monomer and eIF3^nat-40S is required to distinguish the other parts of eIF3 which are not currently well-resolved from the ribosome.

In order to gain further insights into the arrangement of the components of eIF3, its different subcomplexes were reconstituted in vitro, including Tif32/Prt1/Nip1 and Tif32/Prt1/Nip1/Tif34.

These structures will reveal the position of Tif34 and Tif35 (The missing particles) as well as Prt1 (their interacting particle). For defining the position of Nip1, eIF3 was reconstituted with GST-tagged Nip1. GST is a 27 kDa protein which should appear on the EM reconstruction as a blub of density. Knowing positions of Nip1, Prt1, Tif34 and Tif35 one would be able to assign the remaining density to Tif32. Preliminary results in the case of ternary and quaternary complexes have been obtained which indicate differences in density compared to the full complex. However, further analysis is required to confidently determine the differences.