Common principles of protein biosynthesis

A current theory states that it was RNA that stood at the beginning of life on our planet (Gesteland, R.F., Cech, T.R., 1999). Yet, the later appearing proteins outperformed RNA in so many fields that those early, self-sufficient RNA constructs are now extinct. Whereas RNA is composed by a combination of basically four different types of nucleotides and is relatively limited in its ability to form tertiary structure, proteins are highly flexible chains built from twenty amino acids that can fold in a larger variety of three-dimensional shapes. The amount of building blocks (twenty compared to only four in RNA – leaving aside base modifications) equips them with a high degree of adaptability to different tasks and might be the reason for their superiority to RNA in many fields.

To build such a peptide chain, amino acids have to be linked in the correct order via peptide bond formation. Although there exist proteins which are able to catalyze peptide bonds, like the sortase (Mazmanian et al., 1999), in all kingdoms of life the responsibility for building these peptide chains lies in a ribonucleoprotein particle: a macromolecular machine called the ribosome.

General features of the ribosome

The ribosome consists of a large and a small subunit. Both are made up of ribosomal RNA (rRNA) and ribosomal proteins [(]{.FIGURE-HINWEIS}[Figure 1]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS} [(]{.FIGURE-HINWEIS}[Animation 1]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}. The basic mechanism of protein synthesis is very similar in all domains of life: A messenger RNA (mRNA) contains the sequence of the protein and is bound and read by the small ribosomal subunit in collaboration with specific transfer RNAs (tRNAs). tRNAs carry the amino acids and contain characteristic anticodons, which establish base pairing with the respective codons on the mRNA presented to them by the ribosome. Matching allows for addition of the amino acid to the growing peptide chain. After all amino acids have been added, the peptide chain is released from the ribosome and, if needed, further processed by other cell components to become the final, folded protein.

Although the three domains of life, bacteria, eukaryotes, and archaea, all possess ribosomes for protein synthesis, the composition of the ribosomes varies, leading to sometimes very different overall appearance of ribosomes from different kingdoms of life (Amunts et al., 2015; Behrmann et al., 2015; Dunkle et al., 2011; Melnikov et al., 2012; Ramrath et al., 2018) ([Figure 2]{.FIGURE-HINWEIS}

).

The eukaryotic cytoplasmic 80S ribosome is larger than the bacterial 70S ribosome, containing additional RNA segments and additional proteins. Comparison reveals that the eukaryotic ribosome possesses the same conserved structures as the bacterial ribosome in its core ([Figure 2]{.FIGURE-HINWEIS}[A]{.FIGURE-HINWEIS}) and an outer shell where eukaryote-specific elements are located ([Figure 1]{.FIGURE-HINWEIS}[, ]{.FIGURE-HINWEIS}[Figure 2]{.FIGURE-HINWEIS}) (Melnikov et al., 2012).

Not only the ribosomes themselves differ by certain features and are differently sensitive to antibiotics (Yusupova and Yusupov, 2017), but also the interacting factors that render translation possible are for some stages of translation remarkably different (Andersen et al., 2006) and might be an expression of the way the ribosome has been optimized for its bacterial, archaean, or eukaryotic cell environment.

Aim 1

Aim 1

Anatomy

The size of the assembled 80S mammalian ribosome is about 4.3 MDa (Wool, 1979). Both subunits are made of ribosomal RNA (rRNA) and ribosomal proteins (Wool, 1979). Upon joining of the large 60S (50S in bacteria) subunit and the small 40S (30S in bacteria) subunit to the 80S ribosome (70S in bacteria), a functionally important compartment is formed, the so-called intersubunit space [(]{.FIGURE-HINWEIS}[Figure 1]{.FIGURE-HINWEIS}[), ]{.FIGURE-HINWEIS}which is one of the main sites of action during protein synthesis. Across it span three tRNA-binding sites, named A (Aminoacyl)-, P (Peptidyl)-, and E (Exit)-sites. Additionally, the ribosome has specific factor binding sites for the interaction with protein factors, like the P-stalk [(]{.FIGURE-HINWEIS}[Figure 3]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}.

[]{#rRNA is the catalytically active component of the ribosome}

rRNA is the catalytically active component of the ribosome

The rRNA possesses the catalytic activity to perform peptide bond formation and is the main player in protein synthesis. The large (60S) subunit contains three rRNA molecules: the 28S rRNA, the 5S rRNA and the 5.8S rRNA [(]{.FIGURE-HINWEIS}[Suplemental Figure 1]{.FIGURE-HINWEIS}[, ]{.FIGURE-HINWEIS}[Suplemental Figure 2]{.FIGURE-HINWEIS}). The small (40S) subunit contains only one rRNA molecule, the 18S rRNA [(]{.FIGURE-HINWEIS}[Supplemental Figure 3]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}. The rRNA regions responsible for mRNA-recognition, tRNA-binding and peptidyl-transfer are highly conserved in all kingdoms of life (Gesteland, R.F., Cech, T.R., 1999). Among these conserved regions are the sarcin ricin loop (SRL) on the large subunit, interacting with GTP-hydrolyzing protein factors that catalyze certain steps of translation. In the peptidyltransferase center (PTC), also located on the large subunit, the RNA alone is responsible for catalyzing the formation of the peptide bond (Nissen et al., 2000; Spahn et al., 2000). The small subunit 18S rRNA contains the decoding center (DC), which monitors correct tRNA-anticodon matching to the mRNA codon.

Different from bacterial rRNA, eukaryotic 18S rRNA and 28S rRNA contain several expansion segments, long elements of additional rRNA that are to a great deal responsible for the big difference in size between bacterial and mammalian ribosomes. The function of these expansion segments is not yet clear, and their structural investigation is hindered by their high flexibility and peripheral location, making it very difficult to obtain high-resolution structural information (Ramesh and Woolford, 2016; Yusupova and Yusupov, 2017). There is some evidence, however, that expansion segments may play a role in ribosome biogenesis (Ramesh and Woolford, 2016).

Despite the rRNA’s prominent role in translation, the ribosome would not function without ribosomal proteins. They are important for rRNA folding and assembly and stabilize the tertiary structure of rRNA and the ribosome’s functional centers. There are 33 ribosomal proteins on the 40S and 47 ribosomal proteins on the 60S subunit of the mammalian ribosome. Following a recent convention (Ban et al., 2014) universally conserved proteins are prefixed ‘u’, unique bacterial ones ‘b’, unique eukaryotic ones ’e’, and unique archaeal ones ‘a’ [(]{.FIGURE-HINWEIS}[Supplemental Figure 4]{.FIGURE-HINWEIS}[, ]{.FIGURE-HINWEIS}[Supplemental Figure 5]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}.

[]{#The three-dimensional shape of the ribosome is optimized for its function}

The three-dimensional shape of the ribosome is optimized for its function

The 40S subunit can be morphologically divided into several regions, named after the 40S subunit’s resemblance in shape to a bird: Looking at it from the solvent site, on top is the 40S ‘head’ with its prominent ‘beak’, below follows the ’neck’, and the ‘body’ is supplemented by the ‘platform’, ‘shoulder’ and ‘foot’ domains ([Figure 3]{.FIGURE-HINWEIS}). In this work, a rough division into two parts will be used: the 40S head (including beak) and the 40S body/platform, comprising the remaining domains. Importantly, the link between the 40S head and 40S body/platform is flexible and allows for intrasubunit motions. Visible from the intersubunit space, there are the three 40S tRNA binding sites that are distributed among the 40S head and 40S body/platform: A, P, and E ([Figure 3]{.FIGURE-HINWEIS}). The rRNA residues that constitute the tRNA binding sites are well-conserved [(]{.FIGURE-HINWEIS}[Supplemental Figure 1]{.FIGURE-HINWEIS}>, [Supplemental Figure 2]{.FIGURE-HINWEIS}[, ]{.FIGURE-HINWEIS}[Supplemental Figure 3]{.FIGURE-HINWEIS}).

The large (60S) subunit is characterized by several landmarks; The central protuberance, the P-stalk/stalk base and the L1 stalk ([Figure 3]{.FIGURE-HINWEIS}[C-D]{.FIGURE-HINWEIS}). From the solvent side, one can see the ribosomal exit tunnel, from which the newly synthesized protein emerges. The solvent side is to a large degree covered by expansion segment ES7, the largest expansion segment of the 28S rRNA [Figure 3]{.FIGURE-HINWEIS}[C]{.FIGURE-HINWEIS}). Looking on the 60S from the intersubunit space reveals the A-, P-, and E-tRNA binding sites and the sarcin-ricin loop (SRL) ([Figure 3D]{.FH}).

Aim 1

Aim 1

Aim 1

Aim 1

To visualize and mechanistically understand mammalian translocation.

Translocation is one of the least understood processes in protein biosynthesis. Its correct completion is crucial for the continuation of the translation elongation cycle, and ultimately for protein synthesis. As upon translocation, the contacts between the ribosome and the tRNA2•mRNA-module extensively rearrange, it is a process which requires utmost accuracy. Efficient translocation depends on the action of the specialized GTPase eEF2; however, detailed insights into the mechanism, by which eEF2 catalyzes translocation is lacking and opposing hypotheses are vividly discussed: The Brownian ratchet model, in which eEF2 is supporting intrinsic conformational changes that lead to translocation, and the power stroke model, according to which eEF2, being a motor protein, actively moves the tRNAs in the direction of translocation (Chen et al., 2016; Liu et al., 2014; Rodnina et al., 1997; Spirin, 2009).

Moreover, structural knowledge on tRNA translocation is dominated by studies carried out in the bacterial system, and structural data on mammalian tRNA translocation does not exist. Therefore, this work is dedicated to studying how translocation is performed in the mammalian system, using an in vitro reconstitution of a rabbit 80S•tRNA2•mRNA•eEF2•GMPPNP complex. The goal is to obtain structures of mammalian translocation intermediates to characterize mammalian translocation and compare it to translocation in the bacterial and yeast system.

Aim 2

To investigate the 80S•tRNA2•mRNA•eEF2•GDP complex that is observed upon addition of eEF2•GTP to a programmed PRE complex.

To understand why eEF2 can stably bind to ribosomes and two tRNAs in vitro (Budkevich et al., 2014), and how this fits to the model of translocation, I look at an in vitro reconstituted rabbit 80S•tRNA2•mRNA•eEF2•GDP complex.

Aim 3

To revisit the mammalian polysome landscape to find out if serum deprivation influences the distribution of states.

Cryo-EM of actively translating polysomes gives insight into the energy landscape of mammalian translation (Behrmann et al., 2015). Among other stimuli, serum deprivation and subsequent serum restimulation has been hypothesized to influence translation, e.g. via changing polysome patterns (Duncan and McConkey, 1982; Viero et al., 2015). I want to investigate if overnight serum deprivation and 30 minutes of serum restimulation can change the energy landscape of translation.

Aim 4

To look at the phosphorylation site of ribosomal protein eS6 and its possible impact on its surrounding structures to find out the role of eS6 phosphorylation.

The investigation of structural changes induced by eS6 phosphorylation is the fourth aim of this thesis. eS6 is a eukaryote-specific protein of the 40S subunit. It undergoes phosphorylation in response to various stimuli, including serum deprivation/restimulation. Surprisingly, there are almost no works tackling the role of eS6 phosphorylation from a structural perspective. Since structural information on the eukaryotic ribosome from crystal structures and cryo-EM density maps have emerged in the last years, eS6 could be structurally characterized in yeast and recently also in mammalian ribosomes. However, the last residues of the C-terminus of eS6, including all five phosphorylatable serines, are missing in the available structures, and similarly the neighbouring expansion segments are not well resolved, such that the phosphorylation and its structural consequences are not characterized yet (Behrmann et al., 2015; Ben-Shem et al., 2011). This thesis aims to investigate the C-terminal region of eS6 and its surrounding.

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