The translational model for 2A activity suggested that 2A protein may have esterase activity rather than of a proteinase (Ryan et al., 1999). In this model 2A
mediates an attack on the ester linkage between the nascent peptide and the tRNA moiety preventing peptide bond formation between the Glycine residue of 2A and the N-terminal proline of 2B.
During elongation, translocation of the peptidyl-tRNA from the acceptor site (A) into the peptidyl site (P) site occurs, mediated by eukaryotic translation elongation factor 2 (eEF2). This allows the ingress of prolyl-tRNA into the A site. The next step would be formation of the peptide bond between the glycine and proline but nucleophilic attack by the prolyl-tRNA amide nitrogen upon the peptidyl-tRNAgly carbonyl carbon is inhibited by 2A. Thus, prolyl-tRNA is unable to attack the peptidyl(2A)-tRNAgly ester linkage, and the nascent peptide is released by the hydrolysis of the glycyl-tRNA ester bond. The ribosome re-initiates translation of the downstream protein, not with the normal initiator amino acid methionine but with proline.
Translocation Ingress of prolyl-tRNA Translocation Translocation Ingress of prolyl-tRNA Translocation
Figure 15. Proposed model of the mechanism 2A’s action. The nascent 2A peptidyl-tRNA present in the A site (i), is translocated to the P site (ii), leaving the A site free for the ingress of the prolyl-tRNA (iii). The nascent 2A peptide interacts with the ribosome exit tunnel and re-orients the tight-turn at the base of the peptide. This re-orientation of the peptide inside the exit tunnel inhibits the peptide formation between glycyl-tRNA and prolyl-tRNA, leading to the release of the nascent peptide by hydrolysis of the glycyl-tRNA ester bond (iv-v). The ribosome re-initiates translation of the downstream protein with a proline.
Recent studies have shown translation (‘release’ or ‘termination’) factors that are the key for 2A-mediated ‘cleavage’ (Doronina et al., 2008).
Firstly, another line of evidence proved what was already suspected: the 2A reaction occurs in the ribosomal peptidyl transferase centre (PTC) of the ribosome. PTC is formed at the interface between the ribosomal small and large subunits, at the entrance of the ribosome exit tunnel and comprises the activity responsible for peptide bond formation. A series of mRNAs ending at positions spanning the region from glutamic acid at position 14 to the final proline 19 of a 2A were generated and used to program translation reactions assembled with wheat germ extract. Ribosomes stalled at the 3’ end of truncated transcripts, with nascent chains remaining covalently attached to ribosome-associated tRNA. Further, the ribosomal pausing that occurs during translation of 2A is consistent with the hypothesis that the 2A peptide interacts with the ribosomal exit tunnel. Toe-print signals showed that the ribosome pause when the glycine 18 codon (-DxExNPGP-) is at the P site and the proline final codon is at the A site of the ribosome. Replacement of the proline final codon resulted in loss of 2A activity.
It was also shown that translation terminating release factors (RFs) play an essential role in this reaction. Termination of protein synthesis requires two classes of RFs. Class-I release factors (RF1 and RF2 in prokaryotes, and eRF1 in eukaryotes) recognize the three different stop codons (UAG, UGA, UAA) and trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center. Although the function of class-I RFs is similar in prokaryotes and eukaryotes, they exhibit different structural and functional features. eRF1, recognizes all three termination codons, whereas each prokaryotic factor recognizes two out of three stop codons (Scolnick et al., 1968). Class-II release factors (RF3 and eRF3 in prokaryotes and eukaryotes,
respectively) are guanine-nucleotide-binding proteins possessing GTPase activity. eRF3’s activity depends entirely on the ribosome and the eRF1 (Frolova et al., 1996)
and it binds to eRF1. eRF3 can recycle both class-1 RFs (mediated by RF3 in bacteria) and ribosomes (mediated by the ribosome recycling factor (RRF) in bacteria) (Kisselev et al., 2003; Mitkevich et al., 2006). It has also been observed that
RF3 can abort protein synthesis by inducing premature dissociation of peptidyl-tRNA from the ribosome (`drop-off') (Heurgue-Hamard et al., 1998), but the mechanism for
Recent experiments where RF activity was altered revealed an influence in the outcome of the 2A reaction both in vitro and in vivo. Reduced eRF1 levels were
accompanied by reduced synthesis of separated upstream and downstream products, consistent with RF catalysing the hydrolytic termination event. Impaired GTP hydrolysis on eEF3 led to increased production of upstream product and reduction in both extension and downstream products (Doronina et al. 2008). The way this factors
release the protein without recognizing any stop codon is still unknown, but several hypotheses have been suggested.
To date release factors are only known to function at stop codons, and the A site must be empty for them to gain entry into the ribosome. Prolyl-tRNAPro could be unstably bound to 2A paused ribosomes, possibly due to inability to form a peptide bond or, as a second possibility, the ribosomal conformation imposed by 2A could disfavour entry of the tRNA. Prolyl-tRNAPro and RF might compete on ribosomes paused by 2A. However, over-expression of tRNAPro did not reverse the toxic effects of over-expressing 2A in RF-limited cells. Thus the peptidyl(2A)-ribosome interaction and the conformation of the complex disfavours further extension unless RF acts to release the nascent chain or the 2A-ribosome interaction is lost. 2A may also influence and by-pass the necessity for stop codon decoding by RF through directing the ribosome into a conformation similar to that which it takes once RF has bound productively to the A site.
It is interesting to contrast eRF’s action with the well-studied tmRNA system in prokaryotes. tmRNA is a specialized RNA involved in trans-translation, a ubiquitous pathway for removing stalled translational complexes from bacterial cells. Alanine-charged tmRNA, in association with the SmpB protein, recognizes stalled ribosomes, binds like a tRNA to the A-site and donates its alanine to the nascent polypeptide chain in a standard transpeptidation reaction. TmRNA then acts as a surrogate mRNA, replacing the defective mRNA with the self-encoded peptide reading frame, to direct translation (trans-translation) of the degradation tag.
Translation terminates normally at a stop codon provided by the mRNA-like domain of tmRNA. The final translation product of this process carries an 11-residue degradation tag at its C-terminus and thus becomes a substrate for C-terminal specific cellular proteases (Keiler et al., 1996; Yakamoto et al., 2003). This translation quality
knowledge of the control of eukaryotic stalled ribosomes, making possible the involvement of RFs in this case.
All these new findings need to be more extensively studied to find out how the process occurs, however, it is clear that interaction between the ribosome and 2A must exist, as seen in other systems (see section 1.5.1.5), and the presence of RFs add a new piece to the yet to complete puzzle of the 2A mechanism of action.