Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV-IRES RNA

Abstract

The universally conserved eukaryotic initiation factor (eIF) 5B, a translational GTPase, is essential for canonical translation initiation. It is also required for initiation facilitated by the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) RNA. eIF5B promotes joining of 60S ribosomal subunits to 40S ribosomal subunits bound by initiator tRNA (Met-tRNAi(Met)). However, the exact molecular mechanism by which eIF5B acts has not been established. Here we present cryo-EM reconstructions of the mammalian 80S-HCV-IRES-Met-tRNAi(Met)-eIF5B-GMPPNP complex. We obtained two substates distinguished by the rotational state of the ribosomal subunits and the configuration of initiator tRNA in the peptidyl (P) site. Accordingly, a combination of conformational changes in the 80S ribosome and in initiator tRNA facilitates binding of the Met-tRNAi(Met) to the 60S P site and redefines the role of eIF5B as a tRNA-reorientation factor.

Eukaryotic aspects of translation initiation brought into focus

“…The structural details of how eIF5B could concurrently check for both the presence of tRNAi and the sarcin-ricin loop in the context of the large ribosomal subunit had only previously been established at low resolution for the partial bacterial homologue, IF2, which functions in a somewhat different manner [22,23]. Two key cryo-EM structures [24,25], in conjunction with an important crystallographic study of the ribosome [26], have revealed the mechanism by which this takes place. eIF5B is recruited in part by the large subunit, binding in a very similar mode to that adopted by the eukaryotic homologues of the prokaryotic elongation factors, eEF-1 and eEF-2, at the sarcin-ricin loop, in the same position and mode in which these factors that support the addition of successive aminoacyl-tRNA molecules will later bind [24,25].…”

“…eIF5B forms interactions with both the 60S sarcinricin loop and the 40S subunit [24]. This interaction with both subunits stabilizes a rolled conformation of the 40S and 60S subunits within the 80S, which shifts tRNAi towards the peptidyl-transferase centre relative to its position within the 40S initiation complex [25]. Furthermore, the interaction with eIF1A stabilizes the extended carboxy-terminus of eIF5B in a conformation in which it reaches across the A-site, allowing its extreme tip to bind to the tRNAi acceptor stem and orient the CCA-methionine in the correct conformation for incorporation into the peptidyl-transferase centre [24,26].…”

“…Two key cryo-EM structures [24,25], in conjunction with an important crystallographic study of the ribosome [26], have revealed the mechanism by which this takes place. eIF5B is recruited in part by the large subunit, binding in a very similar mode to that adopted by the eukaryotic homologues of the prokaryotic elongation factors, eEF-1 and eEF-2, at the sarcin-ricin loop, in the same position and mode in which these factors that support the addition of successive aminoacyl-tRNA molecules will later bind [24,25]. Binding of eIF5B in this conformation depends on the continuing presence of eIF1A left over after accommodation and the release of the remaining factors, which blocks the A-site to incoming tRNA molecules until subunit joining is promoted by eIF5B.…”

Dynamics of IRES-mediated translation

“…This domain II rearrangement is also present in eIF5B-containing complexes, albeit at an intermediate degree (approx. 41 Å ), where the apical loop positions itself adjacent to the elbow of the initiator tRNA [98] (figure 5c). Domain II was proposed to serve as a 'wedge' by positioning itself to prop open the mRNA channel and widen the P site for tRNA i binding.…”

“…The rolled state is defined as a rotation of the small subunit around its long axis, orthogonal to the previously described inter-subunit rotation [60]. In the case of the 80S : IRES : eIF5B : Met-tRNA i Met : GMPNP complex, the ribosome was classified into two major subpopulations of PRE and POST states, where the PRE state ribosomes were observed as rolled rather than rotated relative to the POST state ribosomes [98]. Thus, the IRES-bound initiating ribosome is conformationally flexible and adopts conformations that are reminiscent of an elongating ribosome, even in the presence of numerous factors and P site-bound initiator tRNA.…”