Inside the ribosome there is an exit channel, which is made of RNA and where the nascent peptides are located. During synthesis this original protein, once it reaches a certain length, enters the tunnel.
It has been shown that this tube is not totally neutral for nascent peptides. There can be some interactions between them that modify the ribosomes action, for example the bacteriophage T4 gene 60 (Farabaugh, 1996; Gesteland & Atkins, 1996) and, also, chloramphenicol (Cm)-resistance genes cat and cmlA (Rogers & Lovett, 1994; Harrod & Lovett, 1994; 1995). T4 gene 60 interacts withthe ribosome, leading to a ribosomal “hop” from codon 47 to codon 50 downstream (Weiss et al., 1990;
Herr et al., 2000). It is also called “translational bypassing” or “subversion of
contiguity” because the determinate gene sequence can control the translation in this way. As has been demonstrated it is essential for the peptide to be a certain length (16 amino acids in T4). Thus the peptide reaches the interior of the tunnel and can interact with the ribosome producing the translational bypass (Gesteland & Atkins; 1996). Another striking example of this nascent peptide-ribosome exit tunnel interaction is the one reported by Nakatogawa and Ito (2002). SecA is a protein involved in peptide export, whose expression is regulated by another protein called SecM, which is encoded in an ORF upstream of SecA ORF. SecM contains an export signal at its N- terminus and an effector motif at its C-terminus, which can block protein elongation stalling the ribosome complex. This occurs only when translocation of SecM through the membrane is impaired by low SecA activity. Thus, the translation initiation region for SecA, which is normally ‘hidden’ by a strong RNA secondary structure preventing initiation of SecA synthesis, is accessible by rearranging of this mRNA secondary structure once the ribosome is stalled. This mechanism creates an efficient intracellular feedback loop for adjusting the supply of SecA proteins to the intracellular demand for protein export. The way this interaction (and others) works is
still unclear but experiments, where mutations where produced in different parts of the ribosome exit tunnel, revealed the interaction to be in the segment of ribosomal protein L22, which is located at the entrance of the exit tunnel (illustrated in figure 16). secM secA SD SD FSTPVWISQAQGIRAGP secA Cell membrane secM secA SD SD FSTPVWISQAQGIRAGP secA Cell membrane secM secA SD SD FSTPVWISQAQGIRAGP secA secA Cell membrane
Figure 16. Representation of the translational control of Sec A by Sec M. Above, illustration of a ribosome with the Sec M nascent protein within the exit tunnel. Below, representation of the Sec M sequence being translated and translocated through the membrane by the Sec A protein. When there is low Sec A, Sec M is able to stall the ribosome, leading to the exposure of the initiation region of SecA, which is normally ‘hidden’ by a strong mRNA secondary structure. In red, shine delgarno sequences (SD). In yellow, effector motif located at the C-terminus of Sec M.
The peptidyl transferase is responsible for catalysis of the transpeptidation reaction in the elongation cycle of translation. In prokaryotes the exit tunnel measures ~80-100Å in length and has a bend some 20-35Å from the peptidyl transferase centre. 2A interacts with the exit channel of eukaryotic ribosomes and this interaction leads to the inhibition of the peptide bond formation between the last amino acid of 2A and the first amino acid of 2B.
The C-terminal –NPG- residues of 2A could play a role in the reorientation of the peptidyl (2A)-tRNAgly substrate to inhibit peptide bond formation and then, stimulate hydrolysis when the A site is occupied by prolyl-tRNA (Donnelly et al.,
2001). The length of the suggested helical part of the 2A sequence is 27Å, which could fit inside the exit tunnel of the ribosome.
This translational model presents a plausible explanation for 2A cleavage activity. First of all a structural model of 2A has been developed that consists of an N- terminal helical portion followed by a tight turn fragment at its own C-terminus (represented in figure 17).
The upstream sequence stabilises and extends the helix and increases interaction with the ribosome exit pore. This α-helix, which interacts with the ribosome exit pore, fixes stereochemistry of tight-turn in peptidyl transferase centre and could have a dipole moment. Inside this fragment there is a glutamate possibly involved in interaction with tRNA and inhibition of peptidyl transferase.
P
PFFFFFF--- ---Q LLNFDLLKLAGDVES NPG
Tight Turn:
• re-orientation of ester bond Tight Turn:
• re-orientation of ester bond
Figure 17: FMDV 2A structural model. 2A sequence is formed by an N- terminal helical portion followed by a tight turn fragment at its own C-terminus.
The highly conserved sequence NPG is proposed to form a tight-turn that reorients the ester bond. Once the peptide bond between proline and glycine (2A fragment NPG) is synthesized, translocation occurs and the 2A peptidyl-tRNA, which was in the A site, is located into the P site. Then, the helical part of 2A interacts with the exit pore and this promotes a specific orientation of the base of the helix within the peptidyl-tranferase centre of the ribosome. Thus, the NPG portion with its tight turn structure reorients the peptide tRNA ester linkage promoting an unusual conformation, different to the usual one that leads to peptide bond formation.
In the model proposed earlier the peptidyl-tRNA ester group would be attacked and thus cut because of the coordination of a water molecule and magnesium ions (Mg2+), which are necessary for the peptidyl-transferase activity. Magnesium ions attach at the base of the 2A helix axis and promote the cleavage in conjunction with a water molecule (Ryan et al., 2004). Thus, when the prolyl-tRNA enters the A
site the formation of the peptide bond is not possible because the usual nucleophilic attack performed by prolyl-tRNA upon the electrophilic centre is hampered. The 2A- peptidyl-tRNAgly ester bond is, then, hydrolysed and the nascent polypeptide released (reviewed by Ryan et al., 2002).
Prolyl-tRNA is the poorest nucleophile among all the aminoacyls-tRNAs since its secondary amino group is sterically constrained due to its specific structure in a five membered pyrrolidine ring.
The suggested model represents yet another trick used by viruses to modify the host cell’s translation apparatus to their own ends. However, as other modifications such as suppression of termination or leaky scanning, this one has not only been observed in picornaviruses but in other viruses and cellular sequences (see section 1.6).