Translation initiation by cap‐dependent ribosome recruitment: Recent insights and open questions

Abstract

Gene expression universally relies on protein synthesis, where ribosomes recognize and decode the messenger RNA template by cycling through translation initiation, elongation, and termination phases. All aspects of translation have been studied for decades using the tools of biochemistry and molecular biology available at the time. Here, we focus on the mechanism of translation initiation in eukaryotes, which is remarkably more complex than prokaryotic initiation and is the target of multiple types of regulatory intervention. The "consensus" model, featuring cap-dependent ribosome entry and scanning of mRNA leader sequences, represents the predominantly utilized initiation pathway across eukaryotes, although several variations of the model and alternative initiation mechanisms are also known. Recent advances in structural biology techniques have enabled remarkable molecular-level insights into the functional states of eukaryotic ribosomes, including a range of ribosomal complexes with different combinations of translation initiation factors that are thought to represent bona fide intermediates of the initiation process. Similarly, high-throughput sequencing-based ribosome profiling or "footprinting" approaches have allowed much progress in understanding the elongation phase of translation, and variants of them are beginning to reveal the remaining mysteries of initiation, as well as aspects of translation termination and ribosomal recycling. A current view on the eukaryotic initiation mechanism is presented here with an emphasis on how recent structural and footprinting results underpin axioms of the consensus model. Along the way, we further outline some contested mechanistic issues and major open questions still to be addressed. This article is categorized under: Translation > Translation Mechanisms Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

“…Furthermore, the observation that this enrichment is significantly more pronounced for eIF2::40Ses than eIF3::40Ses in yeast ( Figure 3L and Figure S10E) indicates that some start codon associated PICs still contain eIF3 but not eIF2. This is consistent with the generally accepted model that eIF2, together with eIF5, must leave the PIC upon AUG recognition, whereas a certain proportion of eIF3 complexes lingers on even during the early elongation cycles (for reviews see (Hinnebusch, 2017;Shirokikh and Preiss, 2018;Valasek et al, 2017)).…”

“…Taken together, our yeast findings obtained with the revised TCP-seq (and their suggested interpretations) largely mirror those found in the original TCP-seq study (Archer et al, 2016), illustrating high reproducibility of this approach. Despite the considerable evolutionary conservation of the initiation mechanism among low and high eukaryotes, mammalian TCP-seq pointed at numerous differences between yeast and humans that probably reflect variations in number of involved proteins, composition of initiation factor complexes and the means of their interactions with 40Ses, as well as with each other (Hinnebusch, 2017;Shirokikh and Preiss, 2018;Valášek, 2012).…”

“…The ultimate purpose of the initiation phase is to bring mRNA to the small ribosomal subunit (40S) in such a way that the start of its coding sequence (CDS) is properly identified by the initiator methionyl tRNA (Met-tRNAi Met ) (reviewed in (Hinnebusch, 2014;Shirokikh and Preiss, 2018;Valášek, 2012)). In eukaryotes, the Met-tRNAi Met is delivered to the ribosome by the translation initiation factor 2 (eIF2) bound to a GTP molecule in the form of a ternary complex (eIF2-TC).…”

Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

“…Which of these two options happens in vivo is not known, but this issue has important conceptual and functional consequences for translation regulation (Jackson et al, 2010;Shirokikh and Preiss, 2018). We reasoned that eIF4Eselective 40S footprinting should allow us to address this longstanding open issue.…”

Deconstructing the individual steps of vertebrate translation initiation

“…Upon recognition of the TIS, the large ribosomal subunit is recruited to form an elongation-capable 80S ribosome. These initiation steps are broadly acknowledged to be a rate limiting factor in protein synthesis 3,5,6 . Despite this, our knowledge of ribosome recruitment, scanning, and TIS recognition is limited.…”