We have discovered that mutations in the apical loop of dII of the HCV IRES inhibit the first translocation event after the formation of the 80S ribosome. HCV IRES-driven 80S ribosome formation and the first step of translocation can therefore be decoupled, a function of dII distinct from previously defined roles of HCV IRES domains. The involvement of dIIb in the first round of translocation could be explained by two broad mechanisms. First, dII’s position in the E site mandates that it move to make way for the P-site tRNA to translocate, evident in cryo-EM reconstructions of the HCV IRES bound to a 40S subunit and 80S ribosome 23,24
. Based on the aforementioned HCV IRES-80S ribosome cryo-EM reconstruction, it has been suggested that contacts between a different portion of dII and the L1 stalk of the large subunit could facilitate domain II displacement from the E site, but this IRES-ribosome contact and potential function has not been tested 24
. Also, a recent crystal structure of HCV IRES domain IIa (not studied) bound to an inhibitor suggest that conformational changes in parts of domain II not studied here may effect movement of the domain from the E site 40
, but again this has not been demonstrated functionally. In the portion of dII studied here, dIIb mutations could potentially inhibit dII displacement, and this would slow translocation by sterically hindering the movement of tRNA. This “failure-to-move” phenotype then could be ascribed to the loss of a specific IRES interaction with rpS5 necessary for dII ejection from the E site, although an analogous role for rpS5 in tRNA ejection has not been reported. This idea does not eliminate the possibility that the L1 stalk and dII conformational changes also help move dII. The second potential explanation is that dIIb actively promotes an event within the ribosome that is important for the first round of translocation, again likely through a specific interaction of dIIb with rpS5 and subsequent conformational changes in the IRES-ribosome complex. Mutation of dIIb would then either interfere with this event, or the IRES would be unable to actively promote this event. These two broad mechanisms are both consistent with our structural and functional data; they are not mutually exclusive.
Given the fact HCV IRES dIIb is positioned to interact directly with the β-hairpin of rpS5, examining known functions of rpS5 (and from bacterial ortholog rpS7) could give insight into how dIIb influences translocation. During elongation, rpS5 (S7 in bacteria) plays a role in maintaining the reading frame and in overall fidelity 37,41,42
. More specifically to the β-hairpin, in bacteria truncation of this structure results in destabilization of the E-site tRNA and an increase in frameshifting and reverse translocation 37
. However, our data shows no evidence for frameshifting induced by the dIIb mutants. Although not explicitly shown to be dependent on rpS5 or S7, it was reported that during elongation the presence of tRNA in the E site influences the fidelity of aminoacyl-tRNA (ac-tRNA) selection in the A site 43
, but other studies find little or no evidence for this 44-46
. Hence, it seems unlikely that the presence of dIIb directly influences entry of A-site tRNA. Overall, we find no known role for rpS5 that readily explains the effect we observed by mutating HCV IRES dIIb, consistent with the notion that the IRES is co-opting this feature of the ribosome to manipulate the complex in a noncanonical way or that rpS5 plays an undiscovered role during translation initiation.
Mutation of dIIb disrupts a putative interaction with rpS5, changes the structure of the IRES-40S subunit complex compared to that of a WT IRES, and inhibits the first translocation step. These observations suggest that a specific rpS5-dIIb interaction induces allosteric changes that propagate through the ribosome. Indeed, there is evidence for a network of interactions within the ribosome that could cause this and also for similar conformational changes induced by bound initiation factors eIF1 and 1A. Specifically, rpS5 interacts with rRNA helices 29, 30, and 42 (refs. 47,48
), and these helices interact with eIF1A (ref. 49
). Another network links rpS5 to the eIF1 binding site through rpS14 and rRNA helix 23 (refs. 41,50,51
). This is noteworthy because binding of eIF1 and 1A induce a conformational change in the 40S that strongly resembles that induced by the WT HCV IRES 23,31
, and eIF1A is known to act with eIF5B after 80S formation to commit the ribosome to elongation 52,53
. This last point raises the interesting possibility that HCV IRES dIIb may induce the same effect as eIF1, 1A, and 5B, to promote a late step during initiation. This notion, speculative at this point, is appealing because a minimal reconstitution of HCV IRES-driven translation initiation does not require eIF1 or 1A 10
, and thus dIIb could substitute for these absent factors. Although not a part of this study, higher resolution structures of mutant HCV IRESs in complex with 80S ribosomes and chemical probing of the rRNA in these complexes before and after translocation could provide insight into the putative allosteric changes associated with this translocation-slowing phenotype.
We would like to propose the following model to explain the role of HCV IRES dIIb in events that occur within the IRES•ribosome complex prior to and during the first translocation step (). First, the IRES assembles an 80S ribosome such that the ribosome is poised at the start codon with an initiator tRNA in the P site. We propose that within this ribosome, dIIb contacts the β-hairpin of rpS5 thereby stabilizing a conformation of the ribosome that is conducive to translocation. Delivery of ac-tRNA to the ribosome by eukaryotic elongation factor (eEF) 1A and subsequent peptide bond formation is then followed by rapid and efficient eEF2-catalyzed translocation. In the case of the dIIb mutants, the mutation induces a local change in structure in the apical loop and this perturbs the interaction with rpS5, affecting 40S subunit conformation. This IRES-40S complex is still capable of progressing to 80S ribosome, but the resultant ribosome’s conformational equilibrium is shifted towards a state with an inhibited ability to translocate. Although ac-tRNA may still be delivered to the A site and a peptide bond formed, the mutant-bound ribosome stalls at the start site. However, because the ribosome samples conformations, these ribosomes are not permanently stalled but occasionally sample a productive state where they are able to translocate. In summary, our data supports a model in which dIIb selects a productive state from the conformational ensemble, while ribosomes bound to IRES with mutated dIIb spend more time in an unproductive state and transition to elongation less efficiently.
Model of domain IIb (dIIb)’s role in HCV IRES translation initiation
Our data open another door to understanding the intricacies of translation initiation and ribosome function. Fundamentally, the ribosome is a Brownian machine sampling many conformations; protein factors and tRNA binding shift the conformational equilibrium, providing efficiency and directionality. Thus, the ribosome is programmed to be manipulated by its binding partners. This inherent characteristic of the ribosome is critical for canonical translation processes and allows subtle and robust regulation of ribosome function. Our results reveal these principles are exploited by a single loop of the HCV IRES, supporting the view of the HCV IRES as a dynamic manipulator of the translation machinery and lending insight into how the translation machinery works in cap-dependent and cap-independent pathways.