Eukaryotic protein synthesis: still a mystery

Much of what is currently understood about eukaryotic protein synthesis represents some very simple principles. DNA makes RNA makes protein. The genetic code accounts for the conversion of nucleic acid information into protein sequence information. The scanning hypothesis and the optimal sequence context around the initiating AUG define most of eukaryotic initiation using the predominant cap-dependent mechanism. The work of many has led to the type of initiation flow scheme shown in Fig. 1. FIGURE 1. The eukaryotic 80 S initiation pathway. A general scheme for the initiation steps in eukaryotic translation (m7G cap-dependent) is depicted. Elements that are uncertain are discussed in the text. This figure is adapted from Fig. 8 in Ref. 8. Despite this detail, this picture is quite deceiving. First, there is more known than is depicted. Recent methodologies of cryo-electron microscopy and high field NMR combined with the almost miraculous crystal structures of bacterial ribosomes have added a considerable amount of information on the specificity and contact (working) surfaces of the ribosome and the translation factors that participate in protein biosynthesis. Second, methodologies are in place that allow for a more detailed analysis of the kinetic properties of the individual steps in this process. Finally, there are more and more examples emerging of elements influential in the regulation of translation as seen as post-translational modifications of the translation factors (or ribosomal proteins), non-coding RNAs, or cis-acting elements in the 5′- and 3′-UTRs2 of mRNAs that recruit trans-acting factors. So where have we gone wrong? To begin with, much of the detail in Fig. 1 was determined in vitro with a series of partial confirmations in vivo, especially in the yeast system. As such, the major limitations to the pathway reflect the fact that most of the evidence used for formulation of the pathway was determined with artificial synthetic mRNAs, globin mRNA, or EMCV mRNA. It is usual to extrapolate from these experiments to more general mRNA translation; however, there are concerns with this approach. All of the in vitro systems function at a rate that is generally 5% (or less) of the in vivo rate, and there is rarely proof of turnover (i.e. that either a translation factor or the mRNA was used more than once). Second, a casual look at the pathway suggests that each translation initiation factor is used once, whereas available data indicate that multiple copies (or rounds of use) are required for one or more of the factors. Finally, each factor used in the in vitro experiments is present in the same stoichiometry. Numbers from mammalian systems and yeast indicate that this is clearly not the case, with the actual concentrations of different factors varying over a hundredfold range. However, the different concentrations of factors could reflect balancing concentration and binding constants or potential multiple turnovers. Thus, although the overall pathway shown in Fig. 1 may be correct, it certainly is not accurate in reflecting the full dynamics and kinetics of the initiation process. Let us begin with “What's Right” with the model pathway shown in Fig. 1. For most mRNAs that are not regulated for expression, this pathway is accurate and reflects the translation of about 80% or more of the cellular mRNAs. Thus, changes that introduce secondary structure into the 5′-UTR reduce expression, as does an alteration of the nucleotide sequence around the initiating AUG (“the Kozak rules”) (1,–3). The reduction in expression due to increased secondary structure can be reduced or eliminated by an increased concentration of eIF4A (4). Although not shown in Fig. 1, translation is very much dependent on the m7G cap at the 5′ end of the mRNA and recognition of this structure by eIF4E. Accessibility to this cap structure is the dominant determinant of the efficiency of an mRNA; this has been shown both experimentally and mathematically (5, 6). This establishes the competitive ranking of mRNAs for translation within the cell. Although not shown pictorially in step 3, this step is the conversion of an RNP complex with secondary and tertiary folding associated with a number of specific and nonspecific RNA-binding proteins into an activated mRNA, the place where the initial competition for translation begins (note that all mRNAs are initially visualized as messenger RNP particles that are about 50% RNA and 50% protein). From the numbers available, most cells contain more mRNA than is being translated, and thus, competition is a common element in translation. A second element that seems correct is steps 7 and 8, which depict the recycling of eIF2 and eIF4F (7). These steps are the major sites for regulation of translation initiation. The control of eIF2 recycling is influenced by either phosphorylation of serine 51 in the α subunit of eIF2 or direct regulation of the activity of the exchange factor eIF2B. Reduction in ternary complexes, eIF2·GTP·Met-tRNAi, as a result of diminishing recycling seems to reduce the translation of all mRNAs equally. By contrast, regulation of the level of eIF4F activity, primarily via 4E-BP, which binds free eIF4E and thereby blocks the reconstitution of active eIF4F, accentuates the competition between mRNAs, more sharply favoring the more efficient mRNAs as the level of eIF4F activity is reduced (8). A third step in Fig. 1 that is also correct is that the selection of the start codon is almost exclusively through the base paring of the anticodon of the Met-tRNAi and the start codon (additional effects via the purine at −3) (9). With this in mind, it then makes perfect sense that the ternary complex should arrive at the ribosome before the mRNA so that no matter where the AUG start codon is, the initiator tRNA would be in position to identify the start codon by base pairing. Where is the pathway in Fig. 1 either incorrect or inaccurate? The pathway is ideally suited for the attachment of the first few ribosomes to an mRNA that begins as a messenger RNP. However, most of protein synthesis is accomplished by polysomes where multiple ribosomes are translating the mRNA. There is thus an apparent kinetic advantage for ribosomes that have just finished translating an open reading frame to undergo initiation on the same mRNA (the circular mRNA). This is envisioned both as a three-dimensional concept (the finishing ribosome is on average closer to the 5′ end of the mRNA than a 40 S subunit free in solution) and as a facilitated process (the interaction between the poly(A)-binding protein and eIF4F, which drives the circularization via protein/protein interactions). A second feature of the mRNA in polysomes is that, at least for the 5′-UTR and the coding region, there is likely very little protein present because it is removed by the scanning process or the process of elongation, although the protein content in the 3′-UTR may be unchanged. The absence of proteins should enhance placement of the mRNA into the mRNA track on the 40 S subunit. Thus, the pathway depicted for translation initiation may be correct for the first few rounds of translation but not for the more efficient rounds of expression from polysomes. Most importantly, the slow steps in the first initiation pathway may be decidedly different from polysome “reinitiation” and thus yield different predictions for the effect of restriction of ternary complexes or active levels of eIF4F.