Architecture and function of ribosomes

Discovery: Ribosomes were first observed in the mid-1950s (PALADE, 1955), since 1958 they are termed ribosomes, due to their composition of a “body” (greek: soma) containing ribonucleic acid (ROBERTS, 1958).

Since 2000 the structure of the prokaryotic 70S ribosome is known (BAN et al., 2000; SCHLUENZEN et al., 2000;

WIMBERLY et al., 2000) and since 2011 the more complex structure of the eukaryotic 80S ribosome is solved (Fig. 2) (BEN-SHEM et al., 2011; KLINGE et al., 2011; RABL et al., 2011), which led to a deeper understanding of processes related to the translation machine. Each ribosomal subunit has its characteristic structural landmarks:

the small subunit displays a body, platform, head and beak, while the large subunit has a more massive, rounded body, with central and lateral protuberances, the acidic stalks (GAMALINDA &WOOLFORD, 2015).

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Figure 2: Structure and architecture of the eukaryotic 80S ribosome. Ribosomal proteins are colored and labeled according to the new nomenclature (BAN et al., 2014). The ribosomal rRNA is shown in grey. Proteins colored in red, orange and yellow belong to the large subunit, proteins colored in blue, cyan and teal belong to the small subunit. If a protein is partially obstructed from view, it may be labeled more than once, even though all ribosomal proteins appear in only a single copy. A) View from the E-site. B) View from the 40S small subunit side. C) View from the A-site. D) View from the 60S large subunit side (adapted from YUSOPOVA &YUSOPOV, 2014).

General features: Ribosomes are ribonucleoprotein particles as they are composed of ribosomal RNA (rRNA) and proteins (r-proteins). They represent central machineries of all living cells that convert the genetic information of the mRNA into proteins during the process of translation. Ribosomes are highly abundant enzymes that can make up ~ 30 % of the total cell mass, and ~ 80 % of the total RNA is rRNA (WARNER, 1999).

Furthermore, prokaryotic cells may possess up to 10⁵ ribosomes which synthesize proteins with an elongation rate of ~15-20 amino acids per second. Eukaryotic cells, in contrast, may contain several millions of ribosomes depending on the cellular state but are characterized by a slower elongation rate of ~5-7 amino acids per second (WEGRZYN & DEUERLING, 2005).

All types of ribosomes are composed of a small and a large subunit that both contain r-proteins and rRNA. Their composition within the different kingdoms of life varies strongly resulting e.g. in a 2.3 MDa ribosome of

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bacteria in contrast to the 4.3 MDa ribosome of human cells (Fig. 3). The functional divergence underlying these structural differences are still not fully understood. Despite these variations the different ribosomal versions share one common core of 34 r-proteins and 3 rRNAs. 4400 bases of the ribosomal RNA are conserved, which harbor the major functional centers of the ribosome as a ribozyme, such as the decoding site, the peptidyltransferase center and the tRNA-binding site. Recent data even describe structural and functional variations of ribosomes within one species, which might change under different conditions of growth or stress (GUNDERSON et al., 1987; MCINTOSH & WARNER, 2007; GILBERT, 2011). In general, the prokaryotic 70S ribosome (3 rRNAs; 54 r-proteins) is composed of a small 30S (16S rRNA; 21 r-proteins) and a large 50S (5S, 23S rRNA; 33 r-proteins) subunit, whereas the eukaryotic 80S ribosome (4 rRNAs, 79 r-proteins) e.g. of Saccharomyces cerevisiae is composed of a small 40S (18S rRNA; 33 r-proteins) and a large 60S (5S, 5.8S, 25S rRNA; 46 r-proteins) subunit (Fig. 3).

Figure 3: Composition of bacterial and eukaryotic ribosomes and the common core. Bacterial and eukaryotic ribosomes share conserved rRNA (light blue) and r-proteins (light red) in addition to their own set of proteins, extensions and insertions in conserved proteins (dark red) and extension segments in ribosomal RNA (dark blue). Dashed lines indicate positions of flexible stalks; for simplicity these lines are not shown in the other structures. The 80S structure of higher eukaryotes has recently be determined (KHATTER et al., 2015). This figure shows a prediction, is highly similar to the yeast ribosome, based on genetic analysis and cryo-EM studies (adapted from MELNICOV et al., 2012).

Subunits and translation: Within the translating ribosome the two subunits fulfill distinct functions: The small subunit is responsible for decoding the mRNA sequence upon initiation of translation as it selects the correct aminoacyl-tRNA. Its major functional regions are the mRNA path, the decoding center and the tRNA binding sites. The A-site serves for binding the aminoacyl-tRNA, the P-site holds the tRNA that carries the nascent polypeptide and the E-site marks the exit of the dissociated empty tRNA. During translational elongation the tRNAs translocate from the A- to the P-site and finally dissociate from the ribosome in the E-site. The large subunit catalyzes peptide-bond formation within the peptidyltransferase center (PTC). Its further functional sites are the tRNA binding sites (A, P and E) as well as the peptide exit tunnel that extends through the body of the

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whole subunit. The PTC is located at the entrance to the polypeptide tunnel in a conserved region on the interface to the small subunit. Upon peptide-bond formation in the PTC, the nascent polypeptide chain is transferred from the peptidyl-tRNA in the P-site to the aminoacyl-tRNA in the A-site, thus extending the nascent chain by one amino acid (aa). Subsequently, the ribosome translocates on the mRNA towards the 3’ end to the next codon and a new aminoacyl-tRNA can bind via its anticodon. During translation the two ribosomal subunits rotate and swivel like a ratchet relative to one another to allow translocation of tRNA and mRNA along the subunit interface (FRANK &AGRAWAL, 2000; HORAN & NOLLER, 2007; ZHANG et al., 2009). The ribosome translates the mRNA sequence from its 5’ to 3’ end into a new polypeptide that is synthesized from its N-terminus to the C-terminus. Upon reaching a stop codon within the mRNA to which no tRNA matches, translation is terminated, the ribosomal subunits dissociate and the nascent polypeptide is released. Translation is assisted by a plethora of factors that provide energy and control the different steps of this complex process. They are termed initiation factors (IFs in prokaryotes, eIFs in eukaryotes), elongation factors (EFs or eEFs), release factors (RFs or eRFs) and recycling factors and differ in their composition and complexity between bacteria and eukaryotes (MELNICOV et al., 2012).

Eukaryotic ribosomes: The eukaryotic 80S ribosome acquired several architecture- and assembly-related features that cannot be found in the structure of the bacterial ribosome. These features include long dynamic rRNA helices on the solvent side of both ribosomal subunits, larger protein clusters assembled round the single-stranded rRNA and unusual interactions mediated by protein tails (MELNICOV et al., 2012; YUSOPOVA &

YUSOPOV, 2014). Remarkably, the interface between both subunits is highly conserved between prokaryotic and eukaryotic ribosomes, indicating that subunit association and the basic mechanism of tRNA recruitment involves conserved mechanisms (SPAHN et al., 2001). Furthermore, the peptidyltransferase active sites show also a high degree of structural conservation, although there are notable differences in the surrounding area (KLINGE

et al., 2011). Many eukaryotic ribosomal proteins, like eL4, uL14, eL22, eL29 or uS3, show unusual folds and contain long tails and loops extending from globular domains. Most of these elongated proteins are not buried within the 80S ribosome but are located at the surface where they can potentially interact with other proteins, likely to control ribosomal function (BEN-SHEM et al., 2011; KLINGE et al., 2011; RABL et al., 2011). Several of these elongated r-proteins interact with long rRNA segments, so-called expansion segments (ES) (MELNICOV

et al., 2012). Some of these segments are tightly associated with rRNAs or r-proteins whereas others comprise long helices that are attached to the ribosome only at their basis, e.g. ES27L (ARMACHE et al., 2010). These types of elongated rRNA segments can adopt different conformations, but the biological relevance of this feature is still not fully understood. In case of ES27L it was suggested that it docks non-ribosomal factors like chaperones or modifying enzymes to the nascent chain that emerges through the ribosomal tunnel exit (BECKMANN et al., 2001).

The ribosomal tunnel: The ribosomal tunnel - through which the nascent polypeptide extends while it is still bound to the peptidyltransferase center - connects the PTC with the cytosol. The tunnel with a length of approximately 80-100 Å and a diameter of about 10-20 Å (NISSEN et al., 2000) is large enough to protect a segment of the growing polypeptide of 30-35 aa in an extended conformation (HARDESTY &KRAMER, 2001) but limits protein folding to the formation of alpha-helices (BHUSHAN et al., 2010). Only in the last 20 Å of the

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tunnel also tertiary structures of the nascent chain might be allowed, as the tunnel widens up in this region (KOSOLAPOV &DEUTSCH, 2009).

In prokaryotes, the tunnel wall is mainly formed by the conserved parts of the 23S rRNA and contains loops of the proteins uL4, uL22 and uL23 (Fig. 4A/B) (BAN et al., 2000; HARMS et al., 2001). In eukaryotes, the area corresponding to the bacteria-specific uL23 overlaps with eL39 (Fig. 4) (BEN-SHEM et al., 2011; KLINGE

et al., 2011). Hydrated polar groups primarily line the tunnel wall that lacks extended hydrophobic patches, which allows passage of all kinds of nascent chains (NISSEN et al., 2000). Together with rRNA the highly conserved proteins uL4 and uL22 form a constriction of the exit tunnel, located ~30 Å from the PTC. This constriction is even narrower in eukaryotic ribosomes, which is suggested to protect the translating 80S ribosome from some macrolide antibiotics that hamper bacterial translation (TU et al., 2005).

Figure 4: The ribosomal tunnel and exit site of prokaryotic and eukaryotic ribosomes. A) Prokaryotic peptide tunnel exit indicated on the slice of the large subunit. Ribosomal proteins involved in the tunnel structure are colored. B) Eukaryotic 60S as in A) C) Structure of the peptide tunnel exit on the solvent side of the 50S subunit. Coloring as in A).

D) Eukaryotic 60S as in C) (adapted from YUSOPOVA &YUSOPOV, 2014).

Until recently the ribosomal tunnel was thought to be inert, however, several studies provide evidence that it plays a more active role in regulating translation and early protein folding. Thus, some proteins interact with the tunnel wall during their synthesis which might regulate translation of the downstream open reading frame and modulate ribosome activity e.g. by the induction of stalling (LOVETT &ROGERS, 1996; TENSON &EHRENBERG, 2002; MANKIN, 2006; HOOD et al., 2009). The tunnel carries an overall negative potential (LU et al., 2007)

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which leads to electrostatic interactions of nascent polypeptides containing elongated stretches of consecutive positively charged amino acids and a transient elongation arrest (LU & DEUTSCH, 2008). In addition to translational regulation, the ribosomal tunnel also provides a defined environment for first protein folding.

Recent data suggest a communication between the nascent chain within the tunnel and exit site proteins that might regulate the recruitment of downstream acting factors (WILSON &BECKMANN, 2011). Taken together, the tunnel represents a universally conserved functional domain of the ribosome, which appears to play a diverse role in protein biogenesis.