Ribosome function

Ribosomes catalyze the fundamental process of translation. In other words, ribosomes translate the genetic information, which is saved in the DNA and delivered by the messenger RNAs, into polypeptides. The process of translation and peptide bond formation is quite conserved in all evolutionary kingdoms (see 1.3.2). In contrast to this, translation initiation differs greatly between Eukarya and Prokarya (see 1.3.1).

translation step Bacteria Archaea Eukarya translation step Bacteria Archaea Eukarya

initiation

IF1 aIF1A eIF1A

elongation

EF-Tu aEF1α eEF1A

IF2 aIF5B eIF5B EF-Ts aEF1B eEF1B (2 or 3 subunits)

IF3 aIF1 eIF1 SelB SelB eEFSec

Table 2. Translation factors in all three evolutionary kingdoms

Orthologous or functionally homologous factors are aligned. (adapted from Rodnina and Wintermeyer, 2009)

1.3.1 Translation initiation

It is not much known about the mechanism of translation initiation in Archaea, but it seems that it is a “mosaic of eukaryal and bacterial features” (Bell and Jackson, 1998). Many eukaryotic translation initiation factors have homologues in Archaea (see Table 2, (Rodnina and Wintermeyer, 2009)), but mRNA binding and start codon recognition is Bacteria-like (reviewed in (Benelli and Londei, 2009)).

In Bacteria (Figure 10 B), all three initiation factors (see Table 2) bind the 30S subunit.

Subsequently the complex binds mRNA, predominantly interacting with the Shine-Dalgarno sequence. The correct positioning of mRNA is achieved by binding of the initiator tRNA in the P site, partially displacing IF3. After subunit joining, GTP is hydrolyzed by IF2 that triggers conformational changes of IF2 itself. This in turn promotes dissociation of initiation factors 12

and accommodation of the initiator tRNA.

In contrast to Bacteria, the initiator tRNA in Eukarya is bound in a ternary complex with eIF2 and GTP (Figure 10 A). The ternary complex together with eIF1, eIF1A, eIF3 and the 40S subunits forms the 43S initiation complex. The modifications of eukaryotic mRNAs, namely a 5'-cap structure (7-methylguanosine) and 3'-polyadenylation, aid the formation of a circular structure. The modifications are bound by the cap-binding protein eIF4E, the scaffolding factor eIF4G and the poly(A)-binding protein (PABP). The 43S initiation complex binds to this structure and subsequently scans along the mRNA for the start codon. During scanning GTP is maybe hydrolyzed by eIF2, but the phosphate is not released until start codon recognition.

The release triggers conformational changes that lead to dissociation of eIF1, eIF2 and eIF5 from the complex and simultaneous joining of eIF5B·GTP. The 60S subunit is binding and upon GTP hydrolysis by eIF5B the remaining initiation factors are released and the initiator

Figure 10. Translation initiation in Bacteria and Eukarya

Canonical translation initiation in Eukarya (A) and Bacteria (B). (modified from Rodnina and Wintermeyer, 2009) (C) IRES dependent translation initiation in eukaryotes. (modified from Jackson et al., 2010) (A)-(C) for details see text

Introduction

tRNA accommodates in the P site.

Interestingly, eukaryotic ribosomes are able to initiate translation independent of mRNA modifications very much like bacterial ribosomes (Figure 10 C, (Jackson et al., 2010)).

40S/43S initiation complexes bind to mRNA secondary structures named IRES (internal ribosome entry sites). These were first identified by analysis of translation initiation of naturally uncapped polio- and picornavirus mRNAs (Pelletier and Sonenberg, 1988; Jang et al., 1988). Up to now, there are four different types of viral IRES-43S/40S initation complex known (see Figure 10 C), which involve different subsets of initiation factors. Remarkably, the type 4 (dicistrovirus intergenic region) IRES dependent initiation is completely independent of any translation initiation factors or initiator tRNAs. Here, an IRES secondary sequence element mimics the tRNA in the P site.

Many non-viral mRNAs have been identified that contain an IRES sequence, though they can also be translated in the canonical, cap-dependent way (Johannes et al., 1999). These include most notably stress response mRNAs and apoptosis related mRNAs (Henis-Korenblit et al., 2000; Mitchell et al., 2001). The key player to switch from cap-dependent to IRES initiation might be eIF4G, which is not only highly over expressed in cancer tissues (Braunstein et al., 2007; Silvera et al., 2009), but in addition its mRNA contains an IRES sequence itself (Johannes and Sarnow, 1998). The translational feedback mechanism of eIF4G expression might shift the translation initiation toward IRES dependent upon high levels of eIF4G and vice versa.

1.3.2 Translation elongation

Translation elongation, as well as termination follows the same principles throughout all living organisms. The tRNAs are bound by an elongation factor/GTP (EF-Tu in Bacteria, for homologues see Table 2) and associate with the ribosome at the A site (Figure 11 A, (Steitz, 2008)), which leads to release of the tRNA from the E site. The correct codon-anticodon pairing triggers GTP hydrolysis and simultaneously the ribosome is subjected to a conformational change that accommodates the A and P site tRNAs in the optimal position for the peptidyl-transferase reaction to occur (see also Figure 6). The transferase reaction itself is catalyzed by universally conserved nucleotides in the 23S/25S rRNA and the tRNA (reviewed in (Beringer and Rodnina, 2007)). The translocation from the pre (A and P site occupied) to the post (P and E site occupied) state is facilitated by binding and GTP hydrolysis of another elongation factor (EF-G in Bacteria).

14

Figure 11. Translation elongation and termination in prokaryotes

(A) Translation elongation cycles in prokaryotes. (B) Translation termination and recycling in prokaryotes.

(A) and (B) for details see text. (modified from Steitz, 2008)

1.3.3 Translation termination and recycling

As soon as a stop codon is positioned at the A site, release factors bind (RF1 or RF2 in Bacteria, for homologues see Table 2). This causes hydrolysis and release of the tRNA-polypeptide at the P site (Figure 11 B). Binding of another factor in complex with GDP (RF3 in Bacteria), followed by exchange of GDP to GTP and subsequent hydrolysis results in dissociation of the release factors and the ribosome. In this post-termination complex (post-TC), the ribosome is still bound to mRNA and a tRNA is left at the P site. The complete dissassembly is facilitated by ribosome release factors (RRF and EF-G in Bacteria). In eukaryotes, this process is conducted by initiation factors (eIF3, eIF3j, eIF1A, eIF1).

Introduction