Where to begin? The mechanism of translation initiation codon selection in eukaryotes

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Selecting the codon at which to begin translation is a complicated event in an already complicated process. Many protein initiation factors (eIFs) have been implicated in start site selection, but the mechanistic details of their activities have remained obscure until recently. Biochemical and genetic studies of eIFs 1, 1A, 2 and 5 have suggested that the 43S pre-initiation complex exists in two conformations and that the changing interactions of the factors within the 43S pre-initiation complex in response to encountering an AUG codon regulates these conformations and, ultimately, the selection of the start codon.

Introduction

The past 40 years of studies of translation initiation in eukaryotes have been synthesized into a model of the steps involved in the formation of an 80S ribosomal initiation complex (Figure 1). First, the eIF2•GTP•methionyl initiator tRNA (Met-tRNAi) ternary complex (TC) is formed (see Box 1 for a list of eIFs involved in start codon recognition). In a process facilitated by eIFs 1, 1A and 3, the TC binds to the small (40S) ribosomal subunit, forming the 43S complex. The eIF4F complex binds to the 7-methylguanosine cap structure on the 5′ end of the mRNA and the factor's RNA helicase activity is thought to facilitate loading of the ribosomal complex onto the message. This process is assisted by eIF3 and the poly(A) binding protein (PAB). The 43S complex presumably scans the mRNA, 5′ to 3′, in search of the AUG start codon in an ATP-dependent process. Usually the 5′-most AUG is selected as the start codon. In mammals, the start codon must also be in a good sequence context, called a ‘Kozak’ sequence, in order to be selected efficiently; the most important bases in this consensus sequence are a purine at position −3 (relative to A in the AUG) and the G at position +4. AUG selection results in irreversible GTP hydrolysis by eIF2 in a reaction promoted by eIF5, a GTPase activating protein (GAP). This is the first irreversible step in the pathway and is thought to commit the complex to beginning translation at the selected codon (as no discard pathway for complexes assembled on the wrong codon has yet been demonstrated). eIF2•GDP is then thought to dissociate, leaving the Met-tRNAi in the peptidyl (P) site of the 40S ribosomal subunit, base paired with the AUG start codon. In the final step, which may occur in concert with eIF2•GDP dissociation, the 60S ribosomal subunit is joined to the 40S subunit containing the Met-tRNAi and mRNA in a process promoted by eIF5B, yielding the final 80S initiation complex awaiting the delivery of an aminoacyl-tRNA to the acceptor (A) site to begin elongation.

The current state of our knowledge of these events has been presented in many reviews encompassing the entirety of eukaryotic translation initiation, from ternary complex formation to subunit joining and beyond [1, 2, 3, 4, 5]. For this reason, the following review takes a more detailed look at a single, critical part of the pathway, selection of the start codon, and concentrates on the recent advances made in elucidating the molecular mechanics that make it possible.

Section snippets

Where to begin?

While all the steps leading to the formation of a translation-competent 80S initiation complex are important, perhaps the most critical of these is the correct selection of the codon at which to begin translation. Incorrect start site selection would at best result in an N-terminally truncated or extended protein, if translation is initiated in-frame, but more frequently would result in the synthesis of a completely miscoded polypeptide. Clearly, mistranslation of the genome to the proteome

eIF1 takes center stage

It is thought that all of the machinery required for the irreversible hydrolysis of GTP in response to start site identification is present in the scanning pre-initiation complex [17••, 20, 21]. Yet, complete GTP hydrolysis is repressed until the start site is found, indicating a change must occur in the 43S•mRNA complex that permits irreversible hydrolysis upon start codon recognition. Several lines of evidence implicate eIF1 as central to this putative change in the 43S•mRNA complex in vivo.

A conformational change and movement of eIF1 upon AUG recognition

Maag et al. recently provided further evidence for a two state, ‘open/closed’ model for the conformations of the 43S initiation complex. By monitoring the stability of eIF1A in various initiation complexes using fluorescence spectroscopy, this study indicated an energetic interaction between eIF1A and eIF5 in the 43S•mRNA pre-initiation complex and implicated this interaction in maintaining the fidelity of start codon recognition [18•]. The interaction between the factors is strengthened upon

The importance of inorganic phosphate

The dissociation of eIF1 from the 43S•mRNA complex after AUG recognition has important implications for start site selection, as eIF1 has been suggested to act as a negative regulator of eIF5-promoted GTP hydrolysis by eIF2 [17••, 26••, 28•]. A recent kinetic dissection of eIF5-promoted GTP hydrolysis by eIF2 has shown that the AUG codon-dependence of GTP hydrolysis is small, only 2–4-fold [29••], consistent with previous results [28•, 30]. The data also suggested that an equilibrium between

A new model

Taken together, the data suggest the following model for start codon selection in eukaryotes (Figure 2). Binding of an mRNA to the 43S complex accelerates a structural rearrangement that makes the complex fully competent to hydrolyze GTP [29••]. One model proposes that this rearrangement is the movement of eIF1 away from eIF2γ (the GTP-binding subunit of eIF2), which allows the N-terminal GAP domain of eIF5 to interact with eIF2γ, resulting in GTP hydrolysis. This model is based on the NMR

Conclusions

Even with the recent advances in elucidating the molecular mechanics of start site selection, many questions remain. What other interactions exist between the components of the pre-initiation complex and how do these interactions affect start site selection? How do previous steps, such as mRNA remodeling and scanning, alter AUG recognition? What are the natures of the different 43S complex conformations? And what within the complex moves during the conformational changes? Although the list of

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Alan Hinnebusch, Sarah Mitchell, Mike Acker, Sarah Kolitz and Julie Takacs for helpful comments on the manuscript. Work in the authors’ laboratory was funded by grants from NIH/NIGMS, American Cancer Society and American Heart Association.

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