Protein synthesis occurs in four stages: initiation, elongation, termination and recycling. In eukaryotes, initiation requires at least 11 initiation factors (eIFs) and can be divided into two steps: formation of a 48S initiation complex and its joining with a 60S subunit (Pestova et al., 2007
). First, eIFs 3, 1, 1A and eIF2•GTP•Met-tRNAiMet
bind to the 40S ribosomal subunit to form a 43S preinitiation complex, which initially attaches to the 5’-proximal region of mRNA after it is unwound by eIFs 4A, 4B and 4F, and then scans to the initiation codon, where it stops and forms a 48S complex with P site codon-anticodon base-pairing. eIFs 5 and 5B mediate subsequent joining of 48S complexes with 60S subunits. During the elongation cycle, elongation factor (eEF) 1A delivers cognate tRNA to the A-site, after which the nascent peptide chain is transferred to the amino acid of the A-site aminoacyl-tRNA. Finally, eEF2 promotes translocation of peptidyl-tRNA from A to P, and of deacylated tRNA from P to E sites. When a stop codon enters the A-site, release factors (eRFs) eRF1 and eRF3 induce hydrolysis of the ester bond of the P-site peptidyl-tRNA (Alkalaeva et al., 2006
). The mechanism of the final step, recycling of eukaryotic post-termination complexes (post-TCs), is completely unknown.
During prokaryotic termination, RF1 and RF2 promote peptide release, whereas RF3 mediates release of RF1/RF2 from post-termination ribosomes, and dissociates after hydrolyzing GTP, yielding post-TCs that comprise 70S ribosomes, mRNA and P site deacylated tRNA (Zavialov et al., 2001
). Recycling of post-TCs requires EF-G, RRF and initiation factor IF3. EF-G and RRF dissociate post-TCs into free 50S subunits and 30S subunits bound to mRNA and P site deacylated tRNA, and IF3 induces release of tRNA from 30S subunits, after which mRNA dissociates spontaneously (Peske et al., 2005
; Zavialov et al., 2005
). RRF, formed by two domains, interacts with segments of 23S rRNA that are involved in forming inter-subunit bridges B2a and B3 (Wilson et al., 2005
). Ribosome binding sites for RRF and EF-G•GTP overlap, and the simultaneous presence of both factors is allowed only if the head domain of RRF is rotated. It was therefore proposed that EF-G•GDP binds RRF-associated 70S ribosomes and exchanges GDP for GTP, and EF-G•GTP induces a rotational movement of the head domain of RRF, which after hydrolysis of GTP by EF-G promotes subunit separation by disrupting B2a and B3 bridges (Gao et al., 2005
Eukaryotes do not encode a RRF homologue, and the mechanisms of the preceding termination stage also differ between the two kingdoms. Thus, whereas RF3 increases the rate of RF1/RF2 dissociation from post-TCs, eRF3 ensures rapid and efficient hydrolysis of peptidyl-tRNA by eRF1 (Alkalaeva et al., 2006
). Binding of eRF1 and eRF3•GTP to pre-termination complexes (pre-TCs) induces their rearrangement manifested as a 2-nt forward shift of their toe-print. However, such complexes are inactive in peptide release, and further rearrangement, induced by GTP hydrolysis, is required to properly position the GGQ loop of eRF1 in the peptidyl transferase center. eRF1, eRF3 and GTP form a long-lived high affinity complex (Pisareva et al., 2006
) suggesting that they likely bind to pre-TCs as an eRF1•eRF3•GTP ternary complex. On the other hand, the mechanism of post-termination dissociation of eRFs is unknown. eRF3•GDP could either potentially dissociate directly after GTP hydrolysis thereby allowing proper positioning of eRF1, or taking into account that eRF1 and eRF3 form a tight complex irrespective of guanine nucleotides, remain bound until the peptide is released and then dissociate with eRF1. The fact that the toe-print shift persists in post-TCs after peptide release suggests that in contrast to prokaryotes, eukaryotic release factors might even remain bound to post-TCs. If this is indeed the case, the mechanism of ribosomal recycling in eukaryotes would likely not be similar to that in prokaryotes, because binding sites for eRF1/eRF3 and prokaryotic EF-G/RRF overlap.
Two observations suggest that eukaryotic ribosomal recycling might not require a special recycling factor, and that initiation factors could mediate this process. First, eIF3, particularly with eIF1, can dissociate 80S ribosomes in the presence of RNAs that can bind directly to the ribosomal mRNA-binding cleft (Kolupaeva et al., 2005
), and could therefore play the principal role in splitting mRNA-containing post-termination ribosomes into subunits. Second, eIF1 can dissociate 48S complexes assembled with initiator tRNA containing mutations in the conserved GC pairs in its anticodon stem (Lomakin et al., 2006
). This activity of eIF1 could potentially be employed to dissociate deacylated elongator P-site tRNAs, because only initiator tRNA contains such GC pairs in the anticodon stem.
We investigated the mechanism of eukaryotic ribosomal recycling using post-termination complexes assembled in vitro on a model mRNA encoding a tetrapeptide followed by a UAA stop codon and report that together, eIF3, eIF1, eIF1A and eIF3’s loosely associated 3j subunit promote recycling of eukaryotic post-TCs.