INTRODUCTION

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Much of the excitement in research on eukaryotic gene expression in recent years has been generated by work on the steps of this process that follow transcription. Taken literally, posttranscriptional gene expression includes all of the steps downstream of transcription that are involved in the realization of the coding potential of the genome, encompassing processes from mRNA modification and processing through to protein folding, sorting, transport, and turnover. However, this review focuses on the fate of pre-mRNA and mRNA during its path through the nucleus into the cytoplasm and its subsequent translation and degradation. In particular, research on the interactions between the translational apparatus and mRNA has uncovered many forms of posttranscriptional control. Moreover, it has become increasingly apparent that many of these different types of control are, in a number of ways, interdependent or coupled to each other.

The yeast Saccharomyces cerevisiae has played a key role as subject and/or host of increasing numbers of investigations in this area and remains a popular organism because of the ease with which it lends itself to genetic manipulation and analysis, in vivo phenotypic analysis, and biochemical experimentation. Moreover, the extensive nature of current knowledge of this relatively simple eukaryote, combined with the attention it is receiving from programs of intensive analysis at the genome, "transcriptome," and "proteome" levels (175, 176, 501, 571), places it first in line for achieving the status of being at least close to comprehensively characterized at some future date. All these points underline the importance of baker's yeast as an organism for study in an area of research like posttranscriptional control, offering, as it does, an increasingly complete picture of how the investigated mechanisms contribute to the physiology and growth of a whole organism. At the same time, it should be remembered that there are aspects of posttranscriptional gene expression that were exclusive discoveries of the yeast research community, including mRNA-specific translational stress responses, mRNA destabilization mediated by upstream open reading frames (uORFs), positive modulation of mitochondrial mRNAs via nucleus-encoded activator proteins, and autocatalytic protein splicing (all of which are discussed in this review).

This review explores the diversity of posttranscriptional control pathways in S. cerevisiae, focusing primarily on those currently known to be mediated or influenced by ribosome-mRNA interactions. Its content and structure reflect the philosophy that the processes of posttranscriptional gene expression should be considered components of a whole rather than being isolated systems. It has therefore been a general aim to examine the interrelationships between the mechanisms underlying posttranscriptional control and their thermodynamic and kinetic consequences at the molecular level. Given that control can be understood only in quantitative terms, the first section sets the stage by considering appropriate theoretical tools for handling control data. In the body of the review, comparisons with analogous prokaryotic and higher eukaryotic systems are made where these highlight key mechanistic principles. Since this review focuses on the issue of control, it does not attempt to serve as a comprehensive compendium of the literature on the cellular components involved. This has inevitably led to the omission of direct citations of many interesting papers, but the reader is encouraged to seek access to these via the cited reviews by other authors.

The rapid development of techniques of molecular biology, biochemistry, genetics, and structural biology over the last few decades has resulted in an explosive increase in the rate of generation of descriptive information relating to cellular components. However, one of the major challenges of contemporary biology is the formulation of physiologically relevant models that describe how these components function in cellular processes. This depends on a successful transition from qualitative to more quantitative representation of living systems which, in turn, requires that biological mechanisms are increasingly described in terms of their thermodynamic and kinetic properties. However, this remains an uneasy interface between the disciplines of the physical and biological sciences, a problem that is exacerbated by the lack of conventions in the use of appropriate terminology. A prime example is the concept of kinetic control as applied to gene expression. As argued previously (361), there already exists a clear definition of control within the framework of metabolic control theory (276, 277), and this provides a suitable basis for unambiguous terminology that can be used to describe posttranscriptional events. This also allows the term regulation to be used in a consistent and unambiguous manner.

Two types of approach to the description and analysis of gene expression pathways will be considered briefly in this review. They offer complementary views that can both be helpful in planning and evaluating quantitative experiments. The systemic approach to metabolic control analysis described by Kacser and Burns (276, 277) was conceived as a means of analyzing complex multienzyme systems which are generally too complex to be amenable to accurate analysis by standard kinetic descriptions of the component reactions. This approach can be usefully applied to the partially processive reactions of pathways such as translation, and some of the concepts are introduced here so that the corresponding terminology can be used later in this review without causing confusion.

The key conceptual tools of the analysis are the coefficients used to define "control" in such a complex system. The most relevant of these in the present context is the control coefficient. This describes the relationship between the activity of each catalytic component (E) and the resulting effect on the flux. The activity variation of each E could be caused by a change in concentration, modulation of its kinetic properties, or the binding of an effector. Each change in a given E component (enzyme) can be expressed as a fractional change, E_i/E_i, and this is reflected in a shift to a new steady-state flux, expressed as F/F. The ratio of the latter to the former represents a measure of the effectiveness of the imposed change in altering the flux, and under the condition E_i  0 it represents a definitive property of the system, called the control coefficient: Z_i = d ln F/d ln E_i. Z_i can theoretically assume any value between 1 and 0: a value near 1 means that E_i has a very strong controlling influence on the overall flux of the system, whereas a value near 0 corresponds to a comparatively minimal contribution to control and probably applies frequently to components of gene expression pathways. An important constraint defined by the Kacser and Burns analysis is the summation principle, which states that the sum of the Z_i values must equal 1. Most importantly, this type of analysis emphasizes that control is distributed among the respective E_is, with the relative individual contributions being reflected in their respective Z_i values. Accordingly, the use of the term "rate-limiting" for any chosen step or E_i in the vast majority of living systems is likely to be misleading and can be meaningless. The Z_i values are determined by a range of factors; in a multienzyme pathway, for example, these factors include the distance of each enzyme from substrate saturation, enzyme concentration, the relationship between the mass-action ratio and the equilibrium constant for each step, and the role of effectors. Analogous properties of the gene expression pathway also contribute to an equivalent set of Z_i values. Unless exceptional forces are at play, there is likely to be selection pressure on a cellular pathway to evolve toward a system in which the Z_i values are not excessively unequal. For example, the provision of certain catalytic components in great excess of their required operational capacities generally makes little sense in terms of cellular energetic housekeeping. Whatever the pathway, this treatment tells us that estimates of Z_i for the respective components are required so that we can model the balance of control.

The above summary of key points intrinsic to the systemic analysis of multienzyme pathways can be seen as a stepping stone to a more consistent theoretical approach to the even more complex pathways involved in gene expression. As will become apparent, although the transition is not entirely straightforward because of the processive nature of at least some of the reactions, the concepts and terminology are useful for even relatively qualitative descriptions of the pathways under examination. For convenience, this review continues to use the term "control" in its generally accepted sense to describe the factors determining the (constitutive) rates of biological processes. However, when applied in discussions of the kinetic details of specific pathways, "control" is applied in the sense explained above and is not intended to imply exclusive rate-limitation by any given step or entity.

The systemic type of model contrasts with the more conventional approximations of pathway kinetics based on several assumptions regarding sites of strong controlling influence (see, for example, references 173, 339, and 340). This second approach is discussed in connection with the specific posttranscriptional pathways as these are addressed in the review. As will become apparent, the latter type of model for a partially processive pathway can be regarded as a special case of the more general approach, but the assumptions on which it is based require careful scrutiny.

One of the least well understood areas of gene expression is how polymerase II (PolII) transcripts are transported from the sites of their synthesis in the nucleus to the sites where they are translated (Fig. 1) (see, for example, reference 404). The significance of this problem in terms of posttranscriptional control can be seen in a number of ways. First, the rate of export to the cytoplasm influences the steady-state availability of translatable mRNA. Second, pre-mRNA and mRNA interact with a range of splicing components and/or heterogeneous nuclear ribonucleoproteins (hnRNPs) (141, 581), most of which have to be effectively replaced at some stage by ribosomes and translation factors in the cytoplasm if translation is to occur. Some hnRNPs shuttle between the nucleus and the cytoplasm (439), and these might be of particular importance to the architecture and translation of the cytoplasmic mRNPs (596). Third, mRNA export and translation may occur simultaneously, possibly with vectorial and energetic consequences for the export process.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Scheme outlining the pathway of eukaryotic transcripts from the nucleus to the sites of translation and decay in the cytoplasm. This review focuses primarily on the posttranscriptional steps of gene expression after nuclear transport. Reproduced from reference 364 with permission of the publisher.

Of these three points, the first two remain at an early stage of characterization and provide us with only a few hints about potential sites of posttranscriptional control on mRNA transport. It is generally agreed that, with very few exceptions, only mature mRNAs (bound by RNA-binding proteins [mRNPs]) leave the nucleus (254, 255). A key feature in this respect is the 5' cap structure. PolII transcripts are capped with methylated terminal structures (516), comprising in S. cerevisiae either m⁷G(5')pppAp (relative frequency, 75%) or m⁷G(5')pppGp (25%). Cap methylation is essential for cell viability (351). The cap promotes mRNA export (198), although experiments with an S. cerevisiae strain containing a temperature-sensitive capping enzyme (Ceg1p, which transfers GMP from GTP to the 5' end of the mRNA) indicate that the cap is not essential for splicing, polyadenylation, or transport (158, 493). In another study, a hammerhead ribozyme was used to catalyze in cis cleavage of a fusion mRNA in S. cerevisiae (144). The capless downstream product was undetectable by standard blotting techniques, as would be expected if the cap is important for nuclear export and/or stability. From the above-mentioned work on CEG1 mutants (158, 493) and further studies described in the section on mRNA stability in this review, it would now seem that the cap influences stability more than transport. At least for histone mRNA, 3' end formation also stimulates the transport process (143).

A heterodimeric nuclear m⁷G cap-binding complex (CBC), comprising two cap-binding proteins (CBP20 and CBP80), has been identified (256), but its role is unclear and it is not essential in yeast (404). In a wider context, discussions about whether nuclear mRNA moves through a "track" (461, 601) or via "channelled diffusion" (612) and discussions about the interactions between mRNA and various nuclear components, including the nucleoskeleton (47), spliceosome components (237, 324, 511, 614, 615), nucleolus (537) and nuclear membrane and/or pore complexes (238), all of which could theoretically influence the transport process, are still under way. To what degree the gene expression processes in the nucleoplasm are structurally or functionally compartmentalized is controversial (505).

The third point raises the issue of how mRNAs find their way to translationally competent ribosomes. Perhaps the most relevant data have come from studies of the Balbiani ring granule, a large RNP particle, in the dipteran Chironomus tentans (367). These results indicate that the particle generally exits the nucleus 5'-end first and is bound by ribosomes before the 3' end passes through the nuclear pore. However, there is no evidence that mRNA export per se requires translation (41). Studies of S. cerevisiae continue to yield new clues about the process of mRNA export. For example, recent evidence indicates that the ATP-dependent RNA helicase Dbp5p, which is a DEAD-box protein closely related to eIF4A, is involved in mRNA export through the nuclear pore complexes (507a, 552a).

While translatable mRNAs are undoubtedly generated primarily by PolII promoters, there is evidence that the pathway outlined above is not the only possible route from nuclear gene to cytoplasmic protein in the eukaryotic cell. Notable in this context is the demonstration that the PolIII promoter of the adenovirus type 2 VA RNAI gene generates uncapped and nonpolyadenylated RNA, which is nevertheless translated, albeit poorly, in HeLa cells (191). This indicates that capping and polyadenylation are not essential for nuclear transport or translation, although it has yet to be determined whether the route taken by such PolIII transcripts may allow them to escape restrictions otherwise imposed on their PolII counterparts. Similarly, it has since been shown that in S. cerevisiae, HIS4 can be transcribed from a PolI promoter to generate primarily uncapped but polyadenylated mRNAs that are poorly translated and rapidly degraded (338a). Overall, these data are in accord with a theme that threads its way through a number of the processes of gene expression: redundancy of function and/or parallel routes provide alternatives for many key steps. What therefore appears to be a major pathway may not be dictated by fixed mechanistic limitations but, rather, may be guided by kinetic or thermodynamic principles of control.

It is clear from the above that there remains considerable uncertainty about the role of any of the nuclear events in posttranscriptional control. Looking beyond the nuclear membrane, there are various lines of evidence for selective distribution of exported mRNA to specific sites within a cell (129, 504, 594) or within whole organisms, for example in Drosophila embryos (504, 589). The fact that mRNA is observed associated with microtubules and actin filaments suggests that these components of the cytoskeleton may be responsible for (selective) mRNA transport (35a). In higher eukaryotes, mRNA partitioning is involved in developmental processes including the establishment of cell polarity and morphogenesis (118, 196, 288) and in the response to signals from the cell surface (89a). Recent evidence also indicates that at least one yeast mRNA (ASH1) becomes localized within the cell by virtue of its association with the cytoskeleton (535). The 3' untranslated region (3'UTR) of this mRNA is necessary for its transport to the distal tip of daughter buds in postanaphase cells. To what extent mRNA sorting plays a role in the yeast cell cycle remains to be established.

Posttranscriptional gene expression begins with a nascent transcript, which goes through a highly complex series of nuclear interactions before emerging into the cytoplasm. However, each transcript is much more than simply an intermediate carrier of genomic coding sequences. A single mRNA can contain several different types of signal element that contribute to one or more forms of posttranscriptional control (Fig. 2). The 5'- and 3'-terminal modifications have a number of general functions that affect the whole mRNA pool. Apart from its role in nuclear export (198), the cap is required for efficient translation (471, 484) and also influences mRNA stability (162), although the extent to which each function overlaps with the others remains unclear. At the 3' end, the mRNA carries a poly(A) tail (initially 60 to 90 adenylate residues in yeast [186, 472]), which also influences the cytoplasmic expression and fate of yeast mRNAs (264, 476). The functions of the untranslated, flanking regions of yeast mRNAs have come under increasing scrutiny in recent years since they, unlike the mRNA modifications, can contain a number of signals that modulate the expression of specific genes in individual ways. Finally, the main reading frame of the mRNA not only constitutes a decodable codon sequence, but also can contain further information in the form of linear signals or conformational blueprints that influence posttranscriptional gene expression, although little is known about them at present. Overall, mRNA carries much more information than merely its coding sequence. The central challenge is understanding how this additional information is able to exert its influence via interactions with the cellular machineries responsible for translation and mRNA turnover.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Features of yeast mRNAs involved in the translation pathway relevant to control. (A) The 5'UTR stretches from the cap to the AUG start codon (positions 1 to 3). (B) Structural features in the 5'UTR that can influence translational efficiency (and mRNA stability) include secondary structures such as stem-loops and poly(G) sequences and short uORFs. uORFs can have a number of important properties, depending on their structure and sequence environment. The main coding region (positions 3 to 5) can sometimes include an in-frame stop codon that either is avoided by frameshifting or, in aberrant mRNAs, leads to premature termination (and mRNA destabilization). The 3'UTR and poly(A) tail (positions 5 to 7) influence the behavior of posttermination ribosomes at the end of the transcript, and at least the poly(A) tail has been implicated in the control of initiation. All of the numbered sites in panel A can be involved in key events of translation or mRNA turnover or act as targets for control mechanisms. The schemes shown are composites of the features of yeast mRNAs that can be involved in posttranscriptional control. Individual mRNAs differ with respect to the combination of the respective sites present. Panel A reproduced from reference 364 with permission of the publisher.

Review articles dealing with the cellular translation apparatus have appeared very recently for mammalian cells (369, 418, 508) and plants (69, 166) and somewhat less recently for yeast (228, 332, 553), and the reader is directed to these reviews for more detailed information about the individual components of the respective systems. In this section I focus on the properties of yeast translation that have most relevance to the known posttranscriptional control mechanisms and how the latest research has shaped the current view of them. The primary pathway of translation in S. cerevisiae is initiated via a cap-dependent mechanism that seems to follow broadly the main pathway that has been delineated on the basis of biochemical studies with mammalian cell extracts. Initiation is not only the most complex step of translation but also a major site for regulation of individual and global gene expression at this level. The subsequent elongation and termination of the polypeptide chain also follow the same general pattern seen in mammalian cells. The overall similarities between the translation machineries of the higher and lower eukaryotes offer the advantage that results gained with both types of system contribute to a general eukaryotic picture of translation. Nevertheless, yeast translation is by no means a carbon copy of its higher eukaryote counterpart, and the increasingly apparent greater or smaller differences between the respective systems can provide additional insight into the structural basis for specific functions in this process. In the following, "yeast translation" refers to protein synthesis in the cytoplasm. However, it should not be forgotten that a very small proportion of yeast proteins are synthesized in the mitochondria.

Before moving on to describe the yeast translational apparatus in more detail, it is useful to consider how the three stages of translation are kinetically related to each other (Fig. 3). Their respective kinetic characteristics are relevant to the functions of the individual components of the translation apparatus; the potential contributions of initiation, elongation, and termination to the control of flux through the whole system; and the interdependency of these three phases. A striking aspect of eukaryotic translation is the processive nature of the events occurring after initiation and the at least partly processive nature of the initiation phase. This has consequences for the ways in which rate control can be exercised on translation (Fig. 3).

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Rate control exercised at different steps of translation. (A) The general scheme indicates the flow rates (j values, in events per unit time for binding and release and in nucleotides per unit time for elongation) assigned to the respective steps of 40S ribosomal subunit binding (jB), 60S junction (jI), elongation (jE), and 40S/60S release (jT). For the purpose of illustrating certain general points, the release rates for 40S and 60S are assumed here to be identical, although this is unlikely to apply to at least some mRNAs. (B) At a low relative rate of elongation (jE1), ribosome packing on the mRNA is high. (C) A reduced packing density occurs at a higher elongation rate (jE2). However, variation in jE need not have a strong effect on overall ribosomal throughput on a given mRNA if the jB and jI rates are not too high. (D) On the other hand, if termination and initiation are coupled, jT may exercise an important control function on translation as a whole.

For example, there are kinetic arguments why initiation can be expected to figure so prominently in terms of posttranscriptional control. Given certain apparently reasonable assumptions, it is relatively easy to arrive at a simplified (nonrigorous) model of the translation pathway (Fig. 3). This scheme summarizes some basic principles concerning potential control points in the protein synthesis pathway. For the sake of simplicity, the steps of translation are represented by flow rates (j values, as defined in Fig. 3). It is assumed that the rate of release of 40S ribosomal subunits from the mRNA during the scanning phase is negligible and that the scanning rate itself is relatively high. The maximal attainable rate of initiation of protein synthesis is dictated by the time taken for the 80S ribosome to clear the AUG region, making space for the next approaching 40S subunit. The region blocked by one ribosome is approximately 30 nucleotides (597); therefore, j_Ij_E/30. The j_E term used here may be adequately described as an average elongation term, but at least in certain mRNAs it may have to be qualified as referring to only part of the open reading frame (ORF). There is evidence that pausing occurs within eukaryotic reading frames, causing ribosomes to "stack," at least in certain regions of the mRNA (597). If termination of protein synthesis (j_T) were to proceed at a much lower rate than initiation (j_I), there would be a blockage that would feed back from the 3' end of the ORF, distorting the structure of polysomes. This is not known to happen (49), but there is evidence for a pause near the termination codon (597), and so it would seem reasonable to assume that for an mRNA with an unstructured leader, j_T  j_I. The scanning component of j_B is unlikely to be slow compared to j_I on unstructured leaders, since this would result in a high loading density of 40S subunits on the 5'UTRs of all mRNAs, for which there is no evidence. Given that pausing seems to occur at the start codon (232, 260, 296, 597), we can assume that j_B  j_I for an unstructured leader. However, the j_B term will be greatly reduced in the presence of structure, thus changing this relationship to j_B < j_I. The above set of principles and assumptions yields a model in which the individual steps of protein synthesis are well matched, allowing the most efficient throughput of ribosomes on an mRNA molecule.

It should be pointed out, however, that while restrictions on j_B, j_I, or j_T can seriously disturb this balance, changes in j_E (at least over a certain range) can be more readily accommodated. In other words, applying the terminology of the Kacser and Burns (276, 277) approach to this processive series of reactions, the control coefficient for elongation is considerably smaller than for the other reactions. A low average rate of elongation (j_E1 [Fig. 3B]) may not greatly affect the relative rate of production of complete polypeptide chains on a specific mRNA unless it results in restricted access to initiating ribosomes (j_I). Increasing the rate of elongation (j_E2 [Fig. 3C]) will also have no effect on the number of polypeptide chains completed in unit time under steady-state conditions unless j_I (and/or j_T) changes. The major difference between the cases in Fig. 3B and C in the steady state will therefore be the density of ribosomal binding. This, in turn, will influence primarily the number of ribosomes bound up in the process of protein synthesis at any one time and hence the availability of free ribosomal subunits in the cellular pool. Where the rate of elongation on individual mRNAs is modulated, for example via the internal nucleotide sequence (codon usage), the impact on the cellular ribosome pool will be negligible, so that the overall steady-state rate of polypeptide production will be relatively unresponsive. Finally, one way of maintaining tight control over the efficiency of the overall process would be to couple termination and initiation, as might occur in the closed-loop model of polysome structure (Fig. 3D).

These are the chief theoretical constraints required to explain how initiation can feature as a step with strong controlling influence (a large control coefficient). The processive nature of elongation means that translation can be represented by a simplified model in which the elongation process is viewed as a single step (i.e., elongation is represented by simply j_E [Fig. 3]). This and other assumptions result in a greatly simplified treatment, which has formed the basis for previous analyses of eukaryotic translation (see, for example, references 173, 339, and 340). One of the questions raised by these analyses is whether the modus of rate control leads to differential attenuation of the translation of individual mRNAs upon shifting a cell from good growth conditions to more restrictive ones. If it is assumed that the poor translation of an mRNA carrying stable secondary structure or a uORF in its leader is based on poor selection of that mRNA by the ribosome, it might be expected that the shift from saturating or near-saturating activity of the translational apparatus to a reduced capacity will affect the poorly translatable mRNAs more severely. However, as discussed below, it is not clear whether selection of an mRNA (i.e., the initial step[s] of initiation) is affected by the structural elements located in the leader. Thus, while the semirigorous analyses reviewed so far bring the issue of control into focus, they do not yet enable us to model translation adequately. This point will be revisited once further information on the translation pathway has been considered.

As discussed later in this review, eukaryotic translational initiation can occur in at least three ways on cellular mRNAs. By far the most common route is the 5'-end-dependent pathway, in which ribosomes apparently select the initiation site via processive scanning along the 5' region of the mRNA. The most striking aspect of the cellular system involved in this pathway is the number and complexity of its components. Apart from the subunits of the eukaryotic ribosome, there are at least 11 eukaryotic initiation factors (eIFs) comprising more than 25 polypeptides (369). These are presented in Table 1. Unfortunately, the disparities between the genetic nomenclatures for the respective organisms makes this subject area highly confusing for the non-specialist. One potential solution would be to adopt a new systematic nomenclature that is related in a readily deducible fashion to the biochemical designations. A proposal of this nature has been made for Schizosaccharomyces pombe which could easily be applied generally (Table 1) (334). Of the yeast initiation factors identified so far, eIF4A, eIF4G, and eIF5A have been found to be encoded by duplicated genes. Like many other duplicated genes in S. cerevisiae, they are at least partially phenotypically redundant (180, 333). Indeed, the eIF4A products are identical. It has been suggested that the maintenance of at least some duplicated genes may reflect past adaptation of the organism to a changing environment (595), but to what extent this type of selective pressure is directly applicable to the initiation factor genes is unknown.

Given the complexity of the initiation process, it has inevitably been easier to characterize partial reactions, each involving a subset of the total pool of eIFs, than to piece together the whole puzzle. The current picture of the pathway derives primarily from experimental work performed on mammalian proteins. The pathway depicted in Fig. 4 has been adapted to take into account the differences so far identified in the yeast system. This undoubtedly simplified version of the real process considers four steps: (i) binding of an active ternary complex to the ribosome; (ii) association of mRNA with the cap-binding complex; (iii) selection of the translational start site; and (iv) initiation of polypeptide synthesis. These steps are discussed in detail below.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Steps of translational initiation in S. cerevisiae. The recycling of eIF-GDP and the suspected dynamics of eIF4F complex formation are indicated schematically. p20 competes with eIF4G for binding to eIF4E, but the means by which this competitive interaction is regulated has yet to be determined. It is now thought to be primarily eIF4F that binds the cap (see next section). The interaction of Pab1p with eIF4G may provide an alternative route to mRNA-ribosome joining, but the significance of such a process in vivo is unknown. In yeast, the relationship between eIF4A-eIF4B helicase/annealing activity and scanning is still controversial. By analogy to mammalian models, other initiation factors have been included in the 43S preinitiation complex that is thought to perform scanning. Many questions remain the subject of further investigation, such as what happens to eIF4F during each cycle of initiation (see Fig. 8) and how eIF3 promotes initiation. It is not known whether scanning is strictly unidirectional. The release of most of the eIFs and the joining of the ribosomal 60S subunit lead to polypeptide initiation. The recent reports on eIF4H (458) and yIF2 (89b), which are not included in this figure, emphasize that we do not yet know the full complement of factors involved in initiation.

Binding of an active ternary complex to the ribosome. eIF2 is a heterotrimeric complex that is required for the binding of Met-tRNA_i and mRNA to ribosomes in vitro. The GTP-charged form of eIF2, which binds Met-tRNA_i in vitro to form the 5S ternary complex, is generated in an exchange reaction catalyzed by eIF2B (45, 480). There is also evidence suggesting that eIF2B promotes the cycling of eIF2-GDP off the ribosome during the initiation process (423a). The component subunits of eIF2 (, , and ) are all essential for cell viability, and mutations in them affect start codon selection by the ribosome (93, 185, 369). Moreover, Cigan et al. (93) also used mutations in the anticodon of one of the four tRNA_i genes in S. cerevisiae to establish the key role of tRNA_i-start codon interactions in initiation site selection. Appropriate compensatory mutations in the anticodon allowed initiation on the HIS4 reading frame by using non-AUG codons that would otherwise not be recognized by the preinitiation complex. The order of the interactions between eIF2-GTP-Met-tRNA_i, the ribosome, and the mRNA is not fully resolved. The bulk of the available in vitro data points to 40S-ternary-complex binding preceding mRNA binding, but Trachsel discusses conditions where this might not apply (552). Certainly, the phenomenon of reinitiation constitutes evidence that close association of 40S subunits and mRNA can occur in vivo before the binding of the ternary complex, at least downstream of a termination event (see below). Finally, earlier this year there was an exciting development in this area. S. cerevisiae was found to possess a homologue of E. coli IF2, called yIF2 (89b). The encoding gene, FUN12, is not essential, but its deletion imposes a severe slow-growth phenotype and a marked translation initiation defect. Biochemical experiments indicate that yIF2, like eIF2, functions to promote Met-tRNA_i binding to the 40S ribosomal subunit (89b). The tantalizing question yet to be resolved is why S. cerevisiae uses both yIF2 and eIF2; does this represent an intermediate stage in ongoing evolution of the translation system in this organism, or are there particular reasons for the maintenance of these parallel functions?

Association of mRNA with the cap-binding complex. eIF4E is the cap-binding component of the initiation factor complex eIF4F, anchoring this complex to the 5' end of capped mRNAs. It is the least variable of the eIF4F components (in terms of presence in the complex and/or protein sequence) and one of the less abundant eIFs (estimated to be present at levels greatly substoichiometric with respect to those of the ribosome [see below]). eIF4E is required for efficient translational initiation in vitro (11), and it is thought that it fulfills the same functional role within the assembled eIF4F complex in vivo. In mammalian eIF4F, eIF4E is associated with two other factors, eIF4G and eIF4A, whereby eIF4G holds the respective factors together in the complex (Fig. 5). Recent reports (121, 168) describe a second human eIF4G gene (encoding eIF4GII), which is 46% identical to the initially cloned gene (eIF4GI [605]) (Fig. 5), and also a second human eIF4E gene, which encodes a protein differing at only two positions. eIF4GII is likely to be a functional homologue of eIF4GI, and there is speculation that the different forms of this protein may be required for a differentiated response to developmental signals (181). The second eIF4E species is likely to be fully functional, and the reason for the duplication has yet to be ascertained. On the other hand, a human protein (4E homologous protein [4EHP]), with 30% identity to eIF4E, has also been described, but as yet it has no apparent function (460a).

View larger version (23K): [in this window] [in a new window]   FIG. 5.   This scheme compares the overall structures and known or predicted binding sites of mammalian eIF4GI (242, 312, 346, 605), its two counterparts in S. cerevisiae (180), and wheat p86 (6, 373). The sites of cleavage by the proteases 2A and L and of the RNA-binding motifs (RNP) are also indicated. The potential eIF3 binding and RNA-binding motifs in yeast proteins have been deduced from sequence comparisons. There is no evidence for the existence of an eIF4A-binding site in the yeast eIF4Gs, whereas there are two such domains in mammalian eIF4G (242). The protein structures are approximately arranged in order to line up the homologous regions. Wheat p86 is thought to have a binding site for microtubules at the C terminus (58). There has been uncertainty about the N-terminal sequence of eIF4GI (181), which is now thought to include a Pabp-binding site (509a). Further examples of eIF4G or of eIF4G-like proteins are discussed in the text.

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View larger version (59K): [in this window] [in a new window]   FIG. 6.   Conserved sequence and structural motifs in eIF4E. Comparison of eIF4E sequences from a range of different organisms reveals the presence of many strictly conserved or conservatively maintained features (boldface type). A number of these are involved in binding the mRNA cap structure (arrows), while others are surface residues (dots), some of which have the potential to be involved in interactions with other proteins (such as eIF4G, 4E-BPs, or p20) (354, 358, 449). Asterisks mark the positions of serine residues in the respective eIF4E sequences that have either been shown or are suspected to be sites of phosphorylation. The sequences shown belong to eIF4E proteins that bind preferentially to the m7GpppX type of mRNA cap. Caenorhabditis elegans has multiple forms of the cap-binding protein (not shown here), at least two of which also recognize m32,2,7GpppX caps (269a). Relatively few differences in the primary sequences apparently suffice to confer this broader specificity on the C. elegans cap-binding proteins.

View larger version (109K): [in this window] [in a new window]   FIG. 7.   Amino acids involved in binding to eIF4G and p20 map to a predicted surface-accessible cluster on the dorsal surface of S. cerevisiae eIF4E (449). Based on the crystal structure of the mouse (27) eIF4E protein (354) and the NMR structure of yeast eIF4E (358), these groups of residues are predicted to lie together on the opposite face of yeast eIF4E from the cap-binding slot. They belong to -helices 1 and 2, respectively, or are associated with a -strand (1) that follows the variable N-terminal region of the eIF4E sequence. The ribbon model shown here is based on the coordinates of the published NMR structure (358). The view is of the dorsal face angled to show the site clearly. The structure of the N-terminal region of the protein is unclear and has been cut off at the top of this representation. The amino acids that affect the binding of the eIF4E-binding domains of eIF4G and of p20 (V71 and W75) are dark grey. The other amino acids seem to influence only binding to the eIF4E-binding domain of eIF4G (E72, H37, P38, L39, and G139).

View larger version (11K): [in this window] [in a new window]   FIG. 8.   Heterotropic cooperativity in eIF4E and the translational initiation cycle. A recent study has suggested testable models that can explain how p20 regulation (A) and cyclical eIF4F function (B) might be achieved (449). Binding of eIF4G to eIF4E induces a high-affinity cap-binding state in eIF4E (A). This promotes 40S-mRNA interactions and ultimately translational initiation. p20 can bind to part of the eIF4G-binding site on eIF4E (Fig. 7), potentially generating a dead-end complex unable to participate in the eIF4G-mediated initiation pathway. Since p20 binds with a lower affinity to eIF4E, it does not block translation but, rather, exerts fine regulation via competition with eIF4G for a shared site on eIF4E. Measurements of the relative binding affinities between these proteins (449) have provided the basis for understanding how a cyclical cap-eIF4E-binding pathway might function (B). The binding of eIF4G mediates both enhanced cap binding and association of the 40S ribosomal subunit. The relatively high affinity of eIF4G binding to eIF4E ensures that the latter binds to the 5' cap almost exclusively as part of the eIF4F complex. Subsequently, and perhaps during scanning or as a result of 60S junction, a rearrangement of the preinitiation complex induces dissociation of eIF4E from eIF4G, which results in the loss of the high-affinity cap-binding state in eIF4E. As a result, eIF4E can be released relatively easily from the mRNA, thus becoming free to rebind eIF4G and thus restart another cycle. Reproduced from reference 449 with permission of the publisher.

Recruitment via the poly(A) tailan alternative route? Recent work has shown that S. cerevisiae eIF4G1 and eIF4G2 also have N-terminal binding sites for the poly(A)-binding protein (539, 541) (Fig. 5). This and other observations (165, 167, 320, 392) have revitalized the debate about the roles of the poly(A) tail and the poly(A)-binding protein in translation (see, for example, the recent review of this theme by Jacobson [264]). Speculation about possible "long-range" interactions between Pab1p and the ribosome had already been stimulated by the isolation of pab1 suppressor strains that harbored mutations in genes encoding 60S subunit proteins (474). Moreover, as discussed below, there is in vitro evidence that the poly(A) tail participates in the recruitment of ribosomes onto mRNAs via a pathway that can function independently of the cap. Investigations in vivo (445, 446, 541), however, paint a more complex picture (see below). The poly(A)-binding protein of higher and lower eukaryotes has four RNA recognition motifs. These contribute to differing degrees to poly(A)-specific and non-poly(A)-specific RNA binding (72, 114, 306, 403, 478). The second RNA recognition motif of Pab1p is required for binding to eIF4G in S. cerevisiae (284), but the actual site has yet to be defined. Most remarkable is the conclusion from mutagenesis studies that Pab1p does not require its specificity for poly(A) to perform functions necessary for cell viability (114). Moreover, while Pab1p binding is shared by wheat eIF-iso4G (320), it has not been clear whether it is also evident in human eIF4GI/II or S. pombe eIF4G (181, 383). There is, however, now agreement that in the former case the site was originally overlooked because of uncertainties about the N-terminal sequence of human eIF4G (509a). Continuing work on S. pombe eIF4G should also resolve uncertainty about Pab1p binding for this fission yeast (449a). A further study has now complicated the story somewhat, since a 480-amino-acid human poly(A)-binding protein (PABP) which shows similarity to the central region of eIF4G has been described (102). The results of continuing investigations of these various Pab1p-related proteins and interactions are awaited with interest.

Selection of the translational start site. The prokaryotic ribosome can locate a start codon via direct interactions with sequence elements such as the Shine-Dalgarno region located within a translational initiation region (TIR) that has evolved to guide and modulate the initiation process (177, 178, 362, 363). In contrast, despite the theoretical proposition that "translation-initiation promotion sites" may enhance the expression of certain genes (545), there is no experimental evidence for such rRNA-mRNA interactions mediating start site selection in the eukaryotic cell. Instead, initiation on the vast majority of cellular mRNAs involves a process currently modelled by the "scanning hypothesis" (to be considered in more detail below). Investigations of mammalian in vitro systems have indicated that the process of scanning through structural leader regions to the start codon may be driven by the helicase activity of eIF4A (which is enhanced in association with eIF4B), possibly associated with the small ribosomal subunit (509). eIF4A belongs to the DEAD-box family of proteins, which possess ATPase and ATP-dependent RNA helicase activities (161, 487). It is required for translation in a yeast cell extract (57). However, it is not included in the eIF4F complex isolated from S. cerevisiae, and it is therefore questionable whether it cycles through the yeast eIF4F complex, as has been suggested for the mammalian protein (420). On the other hand, it seems unlikely that the function of eIF4A is linked solely to its potential role in destabilizing secondary structure in the 5'UTR, since in the experiments of Blum et al. (57) it was required for the translation of an mRNA that had a relatively short, unstructured leader. Overall, the role of the RNA helicase activity of eIF4A and eIF4B in scanning remains poorly defined.

Initiation of polypeptide synthesis. Once the 40S subunit has located a start codon, the 60S subunit joins it to form the 80S initiation complex, which can then begin with peptide bond formation between the initial methionine and the second encoded amino acid. This ribosome-joining step is promoted by eIF5, which, as we have seen, acts to ensure the fidelity of the Met-tRNA_i-start codon interaction (85, 236, 369). A number of other factors are likely to be released at this point. Of particular interest is the dissociation of eIF2, which is now complexed with GDP after hydrolysis of the GTP that was originally bound and which will have to be recycled back to the GTP form by eIF2B in preparation for a further round of initiation. The mechanism of this GDP-GTP exchange reaction is still the subject of lively discussion (552).

Additional factors involved in translation. There is quite a collection of proteins that, like Pab1p, seem to be involved in, or influence, the initiation process but are not formally classified as eIFs. This is something of a grey area in terms of formal classification, but it may also be indicative of important but as yet uncharacterized functional interactions between the translational apparatus and other cellular components. For example, Donahue and colleagues (190, 609) isolated four unlinked genes (SSL1 to SSL4) which, when mutated, can suppress the inhibitory effect of a stem-loop structure in the 5'UTR of HIS4. SSL1 and SSL2 were more recently determined to encode components of the transcription factor TFIIH (578). SSL2 was also found to be a yeast homologue of the human ERCC-3 gene, thought to be involved in DNA repair (190). It is still unclear to what extent the observed effects of the SSL mutants on translation reflect the normal roles of these genes in wild-type cells. SIS1, which encodes a yeast homologue of Escherichia coli DnaJ, has also been linked to translation (617). The temperature-sensitive phenotype of a SIS1 mutant was suppressed by one of the ribosomal gene deletion mutants that was previously shown to suppress a pab1 mutant (474). Again, while links between translation and other cellular processes are suggested, the mechanism underlying this effect remains unknown. It is interesting that the theme of chaperones apparently influencing translation is also evident in the phenotypes of mutations in two genes encoding 70-kDa heat shock proteins (hsp70s), i.e., SSB1 and SSB2. In this case however, the translation defect was suppressed by overexpression of the HBS1 gene, which encodes a protein resembling eIF1A and eRF3 (401). The Ssb proteins may be core components of the translating ribosome, perhaps preventing misfolding of nascent polypeptide chains (436a). Finally, one additional factor has been found that seems to be required for translation in wild-type yeast cells (91, 119). This is Ded1p, which is a DEAD-box protein originally identified as a potential suppressor of defects in pre-mRNA splicing (269) and PolIII (549). Ded1p is required for translational initiation in vitro (91), and ded1 mutants are defective in translational initiation (91, 119).

Reaction pathways and kinetic control. While it is convenient to break down the initiation process into distinct steps, there is in fact little information on the spatial and temporal relationships between the respective partial reactions within the living cell. For example, the translational apparatus can hardly be envisaged as a farrago of randomly acting components, but is there a highly ordered multifactor supercomplex (a "translatosome"), or does the reality lie somewhere between these two possible extremes? It is impossible to resolve issues such as this on the basis of in vitro experiments alone, since disruption of the complex and delicate pathways in the cell may leave only partially or completely uncoupled component reactions. Effectively torn out of its natural cellular environment, translation in vitro is likely to reflect, but unlikely to reproduce, the bona fide process in vivo. This problem may be particularly applicable to the initiation step, since this encompasses not only the transition from the nuclear phase of the life of an mRNA to its recruitment by the translational machinery but also the orchestration of the most complex phase in protein synthesis. Also linked to this problem is the fact that initiation is not an autonomous process occurring independently of the other phases of translation. Statistically, it would be expected that most ribosomes initiating protein synthesis on an mRNA had recycled after previously terminating at least one other polypeptide chain. In general, this means that the pool of ribosomes available for initiation is subject to control by the termination process and posttermination events. Moreover, there are theoretically two extreme cases where this control might be exercised within the confines of a single polysome: as a consequence of reinitiation on a multicistronic mRNA (see below), and in the context of a "closed-loop" (265) type of polysome structure which might allow a ribosome terminating at the 3' end of the mRNA to be recycled back to the 5' end (see, for example, reference 233) or to influence de novo initiation at the 5' end. Reinitiation is known to occur (see below), while the latter type of mechanism is feasible but has not been confirmed as a potential pathway. These considerations emphasize the importance of the cyclical nature of the operations performed by the translational machinery and thus of the relationship between initiation and the other phases of translation.

Elongation factors. The process of elongation in eukaryotic translation has generally received comparatively little attention and is assumed to function in an analogous fashion to that of its counterpart in E. coli (459). While this assumption is certainly likely to apply to the basic biochemical principles, the eukaryotic systems have their own, more complex, set of elongation factors. A highly abundant homologue of the bacterial factor EF1A (formerly called EF-Tu) is present (eEF1A). This forms a ternary complex with GTP and aminoacyl-tRNA and promotes binding of the latter to the ribosomal A site. The other eukaryotic factors (eEF1B and eEF2) do not show readily identifiable sequence homology to their prokaryotic counterparts (EF1B and EF2, formerly known as EF-Ts and EF-G, respectively). The heterotrimeric factor eEF1B catalyzes GDP-GTP exchange on eEF1A yet is dissimilar to the prokaryotic factor EF1B (EF-Ts), which performs an analogous function for EF1A. However, in yeast, eEF1B is rendered redundant if eIF1A is overexpressed (286). The other G-protein, eEF2, is thought to be required for translocation of the peptidyl-tRNA to the P site and, by analogy to the prokaryotic system, of deacylated tRNA to the E site. eEF2 is potentially a major site of regulation mediated by phosphorylation in higher cells (395, 418). An intriguing property of the eukaryotic factors eEF1A and eEF2 is their ability to bind to cytoskeletal components (36, 500), since this may provide a mechanism for the intracellular transport of mRNA, perhaps within polysomes. eIF1A and eEF2 are encoded by duplicated genes. In both cases, the encoded proteins are identical whereas there are only minimal differences in the respective reading frames (99, 394, 433).