It comprises four phases: initiation, elongation, termination, and recycling Fig. The initiation phase involves the binding of the small ribosomal subunit 30S to an unstructured region in the mRNA the Shine-Dalgarno region that is complementary to a portion of the 16S rRNA, and is stabilized through an interaction between the ribosome-bound initiator formyl-methionyl-tRNA and the initiation codon, usually AUG.
The large ribosomal subunit 50S then binds to form a 70S initiation complex, which contains the formyl-methionyl-tRNA in the ribosomal P-site and which is prepared to enter the elongation phase. These reactions are promoted by a number of elongation factors and by the ribosome itself, with rRNA playing a particularly notable part. Ribosomes are then recycled through interactions with a number of protein factors to generate ribosomal subunits capable of undergoing another round of protein synthesis.
Detailed descriptions of the bacterial pathway are found in a number of reviews Laursen et al. Pathway of protein synthesis in bacteria.
Not shown are the initiation, elongation, and termination factors that promote the reactions. Following initiation, each turn of the elongation cycle results in the addition of another amino acid gray pentagon to the growing peptide chain not shown. Protein synthesis in higher cells shares many similarities with that in bacteria.
The genetic code is identical and the aminoacyl-tRNAs and their synthetases are very similar, but eukaryotic ribosomal subunits, named 40S and 60S, are larger and richer in protein, as illustrated by recent high-resolution structures see Wilson and Cate A conspicuous feature of eukaryotic protein synthesis is the fact that mRNAs are translated in the cytoplasm, making translation uncoupled from transcription.
They are generally monocistronic, unlike most bacterial mRNAs, and the pathway and mechanism for the formation of 40S and 80S initiation complexes differ substantially from those in bacteria. Therefore 40S initiation complex formation involves numerous protein—RNA and protein—protein interactions, in contrast to what occurs in bacteria.
Given the preeminence of the initiation phase in the regulation of protein synthesis, we develop the mechanism of eukaryotic initiation in considerable detail in the following section.
Termination and recycling resemble the reactions in prokaryotes, except that different sets of proteins promote these phases. Eukaryotic initiation pathways are outlined in Figure 2 ; detailed descriptions of the molecular mechanisms are found in Hinnebusch and Lorsch Pathway of eukaryotic initiation. Not shown are the initiation factors or the possibility that scanning follows IRES-directed binding of the 40S ribosomal subunit during internal initiation.
To elucidate translational control mechanisms, it is essential to define the detailed molecular mechanism of protein synthesis. The 60S ribosomal subunit then joins the 40S initiation complex to form an 80S initiation complex capable of entry into the elongation phase.
These reactions are promoted by twelve or more initiation factors comprising over 25 proteins see Hinnebusch and Lorsch Although much has been learned about how mammalian cells initiate protein synthesis, a number of gaps and uncertainties remain. For example, identification of initiation factors has been based on their stimulation of in vitro initiation assays constructed with purified components, and verified by genetic methods.
However, the recent discoveries of new proteins apparently involved in the pathway e. In addition, the relevance of some identified factors is uncertain. Despite its centrality, aspects of the scanning mechanism are not yet well elucidated. Are there mechanistic clues in the unusual bidirectionality of the helicase activity of eIF4A, and the departures from stoichiometry in the levels of some of the factors? Because rigorous kinetic analyses of many of the reactions in initiation have not been performed, we do not have a full description of their reaction rates, yet such information is essential for detecting and understanding regulation during initiation.
In contrast, great progress has been made recently in elucidating the structure of eukaryotic ribosomes see Ben-Shem et al. Still other initiation pathways have been described, involving shunting Yueh and Schneider ; Pooggin et al. Although we already possess much sophisticated knowledge of how these initiation pathways proceed, there remains much to be learned that is essential for a full understanding of translational control.
Regulation of protein synthesis may occur at different steps of the pathway, with the initiation phase being the most common target. Which phase of protein synthesis is affected is often identified by determining polysome profiles Merrick and Hensold and ribosome transit times Fan and Penman ; Palmiter One of the salient features of translational control involves the number of mRNAs affected. Global regulation often is based on the activation or inhibition of one or more components of the translational machinery, whereas specific regulation frequently occurs through the action of trans -acting proteins see Gebauer et al.
Some mRNAs are capable of escaping the effects of global activation or inhibition. Therefore, the response caused by a given mechanism may be complex in terms of the mRNAs affected. Methods that address this latter issue are ribosome profiling Ingolia et al.
Such analyses of changes in ribosome profiles caused by a difference in physiological state enable identification of the mRNAs that are most affected. The polysome profiling method is particularly powerful, as it determines where ribosomes are positioned on essentially all cellular mRNAs at a specific point in time, thereby shedding light on the phase of protein synthesis that changes. Another important feature of translational control is that a change in physiological state can activate multiple regulatory mechanisms that affect the rate of protein synthesis.
Such redundancy complicates mechanistic studies, because interfering with one mechanism does not necessarily alter the overall extent of inhibition or activation. A further complication is that a given mechanism may itself cause only a minor change in protein synthesis rate.
However, when multiple weak mechanisms act on the system together, significant translational control can result. Mechanisms that are modest in their action are especially difficult to elucidate, as their effects sometimes only slightly alter a specific reaction rate. To detect and assess the importance of such mechanisms, sophisticated and highly accurate kinetic analyses are required, and are increasingly being pursued.
Recruitment of the mRNA to the 40S ribosomal subunit is thought to be the rate-limiting step of initiation, and is often modulated. The binding of methionyl-tRNA i also is frequently regulated, and subsequent steps such as scanning and initiator codon recognition may be affected as well. How are these reactions regulated? The cellular levels of the canonical initiation factors differ in various cell or tissue types, thereby affecting initiation rates.
Modulating the activities of the initiation factors by phosphorylation is often used to regulate global rates of protein synthesis. Numerous other initiation factors are phosphorylated, often as targets of signal transduction pathways, as are ribosomes and the elongation factor eEF2, but how such events regulate protein synthesis is not yet well established.
Besides phosphorylation, posttranslational modifications such as methylation, ubiquitination, and glycosylation, may affect protein synthesis, but these have not been studied extensively. One can anticipate that mass spectrometric methods will identify new modifications of importance in the near future. Regulation of translation through the action of microRNAs is an exciting new area of study. Precise mechanisms whereby the microRNAs affect protein synthesis are yet to be elucidated.
The presence of one CPE close to, but not overlapping with, the hexanucleotide sequence AAUAAA was shown to promote early polyadenylation, and thus to stimulate translation, while the presence of a cluster of CPEs in which one overlapped the AAUAAA sequence or was separated from it by only one to three nucleotides promoted repression of translation and late polyadenylation.
Sequence elements in UTRs can also enhance translation. But Harris showed that, under conditions in which cap-dependent translation is inhibited, cap-independent translation occurs, and that this mechanism is distinct from IRES-driven activity and requires both the 5' and 3' UTRs.
This shows for the first time that translation initiation in a viral system can switch from being cap-dependent to cap-independent, a property previously described only for cellular mRNAs. RNA-binding proteins have key roles in the regulation of nearly every aspect of gene expression. They often display a modular architecture, exert multiple functions and participate in more than one step of the gene-expression pathway.
He showed that binding of ZBP1 probably inhibits translation by interfering with the formation of the 80S ribosome during initiation. Two different pathways for phosphorylating ZBP1 are involved in this regulation. In addition to being part of the translation machinery, some ribosomal proteins have roles in translational control. This complex binds to a previously identified stem-loop structure in the 3' UTR of ceruloplasmin mRNA, and is responsible for silencing its translation in macrophages associated with inflammation.
Fox showed that, when L13a is phosphorylated, it is released from its site in the 60S ribosomal subunit to form, with three other proteins, the functional GAIT complex that binds to ceruloplasmin mRNA and blocks its translation. RACK1 also called Asc1p in Saccharomyces cerevisiae acts as a scaffold for recruiting proteins involved in signaling pathways and has been linked to the control of translation initiation of specific mRNAs.
RACK1 appears to expose a platform surface to the solvent, possibly providing a scaffold for interacting factors. The location and shape of RACK1 are also conserved in yeast ribosomes, indicating its general role in linking signal transduction pathways to the ribosome.
Translational control by cytoplasmic polyadenylation is crucial not only for development but also for regulation of 'synaptic memory' in the mammalian central nervous system. Long-term synaptic plasticity and long-term memory require the synthesis of new proteins for their consolidation. The signaling pathways that are responsible for initiating new protein synthesis are poorly understood, but most regulation is thought to take place at the level of translation initiation.
Mice with a genetic knockout of eIF4E-BP2, a factor that inhibits translation, showed altered long-term synaptic plasticity and deficits in long-term memory. Several post-transcriptional mechanisms are used by eukaryotic cells to control the quality of mRNA.
It is affected by many factors such as sex, hormones, cell cycle, growth and development, health status and living environment, as well as the changes of many biochemical substances involved in protein synthesis. Because translation and transcription in prokaryotes are usually coupled and their mRNA lives are short, the speed of protein synthesis is mainly determined by the speed of transcription. Weakening is a way of regulating the speed of translation by affecting transcription in the first place through the excess and inadequacy of translation products.
The structure and properties of mRNA can also regulate the speed of protein synthesis. The initiation of translation is that eIF-3 binds to 40S subunit and promotes the dissociation of 80S ribosome from 60S subunit.
Compared with prokaryotes, the marked difference in elongation of eukaryotes lies in their separation from transcription. Although prokaryotes can undergo two cell processes simultaneously, the spatial separation provided by the nuclear membrane prevents this coupling in eukaryotes. Eukaryotic elongation factor 2 eEF2 is an adjustable GTP-dependent translocation enzyme that moves a new polypeptide chain from site A to site P in the ribosome. Translation of mRNA into protein represents the final step in the gene-expression pathway, which mediates the formation of the proteome from genomic information.
The regulation of translation is a mechanism that is used to modulate gene expression in a wide range of biological situations. From early embryonic development to cell differentiation and metabolism, translation is used to fine-tune protein levels in both time and space 1 , 2. However, although many examples have been described, much remains to be learned about the molecular mechanisms of translational control.
Two general modes of control can be envisaged — global control, in which the translation of most mRNAs in the cell is regulated; and mRNA-specific control, whereby the translation of a defined group of mRNAs is modulated without affecting general protein biosynthesis or the translational status of the cellular transcriptome as a whole.
A special, and extremely interesting, case of mRNA-specific regulation is the local regulation of translation that occurs in a polarized cell.
The translation of specific mRNAs is restricted to defined locations, such as the anterior or posterior pole of an oocyte, or a specific neuronal synapse. The purpose of this regulation is to generate protein gradients that emanate from a particular position in the cell, or to restrict protein expression to a small, defined region — for example, to a synapse. Although such local translational control almost invariably involves regulatory complexes that associate with the target transcripts, it might also use local changes in the activity of general translation factors 3.
Structural features and regulatory sequences within the mRNA are responsible for its translational fate Fig. Although, in principle, regulation could activate or repress translation, most of the regulatory mechanisms that have been discovered so far are inhibitory, which implies that, unless a regulatory mechanism is imposed, the mRNAs are translationally active by default.
However, this does not mean that all non-repressed mRNAs are actively engaged with ribosomes, because the activity of translation-initiation factors, particularly those that support the recruitment of ribosomal complexes that initiate translation, is frequently limiting. As a consequence, most mRNAs are distributed between an actively translated and a non-translated pool in the cytoplasm of cells, and changes in the activity of these limiting translation factors elicits changes in global protein synthesis.
Secondary structures, such as hairpins, block translation. Internal ribosome entry sequences IRESs mediate cap-independent translation. Upstream open reading frames uORFs normally function as negative regulators by reducing translation from the main ORF. In this review we will discuss the detailed molecular mechanisms of translational regulation, by focusing on examples of both global and mRNA-specific translational control. The translation process can be divided into three phases — initiation, elongation and termination.
Whereas the elongation and termination phases are assisted by a limited set of dedicated factors, translation initiation in eukaryotes is a complex event that is assisted by more than 25 polypeptides 4 , 5 , 6. Translation initiation involves the positioning of an elongation-competent 80S ribosome at the initiation codon AUG. The large 60S ribosomal subunit then joins the 40S subunit at this position to form the catalytically competent 80S ribosome Fig.
Here we provide a succinct overview of the process of translation initiation as far as it is directly relevant for the examples of translational regulation that are discussed below. For a more detailed description of the translation-initiation process, see Refs 4 — 6. Only the translation-initiation factors that are discussed in the main text are depicted; others have been omitted for simplicity.
Eukaryotic initiation factors eIFs are depicted as coloured, numbered shapes in the figure. For a complete account of translation-initiation factors, see Refs 4 , 6. This complex then binds to the small 40S ribosomal subunit, eIF3 and other initiation factors to form the 43S pre-initiation complex. Subsequent joining of the large 60S ribosomal subunit results in the formation of the 80S initiation complex.
Subsequently, the 80S complex is competent to catalyze the formation of the first peptide bond. P i , inorganic phosphate. The small ribosomal subunit, together with other factors, forms a 43S pre-initiation complex that binds to the mRNA. However, as direct physical evidence for scanning intermediates remains to be found, scanning is probably a rapid process that involves unstable intermediates.
Translation initiation requires energy in the form of ATP. However, scanning of unstructured leaders can occur in the absence of ATP in vitro , which indicates that the movement of a 43S complex along the mRNA might not require energy unless the ribosome encounters a stable structure in the mRNA Although it is unclear at present whether the 43S complex remains physically associated with the cap structure during scanning, the eIF4F complex has been shown to support scanning Binding of the 43S complex to the initiator codon AUG results in the formation of a stable complex, which is referred to as the 48S initiation complex.
Selection of the correct initiation codon critically depends on eIF1 Refs 11 , The 43S complex recognizes the initiation codon through the formation of base pairs between the initiator tRNA and the start codon. Subsequently, eIF2-bound GTP undergoes hydrolysis that is catalyzed by eIF5 — a reaction that is necessary, but not sufficient, for the 60S ribosomal subunit to join the initiation complex.
Global control of protein synthesis is generally achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them. Two well-characterized examples are discussed here. As mentioned above, eIF2 is part of the ternary complex and associates with the small ribosomal subunit in its GTP-bound form. These include: the haem-regulated inhibitor HRI , which is stimulated by haem depletion; GCN2 general control non-derepressible-2 , which is activated by amino-acid starvation; PKR protein kinase activated by double-stranded RNA , which is stimulated by viral infection; and PERK, which is activated under circumstances of endoplasmic reticulum ER stress.
The availability of the cap-binding protein eIF4E is also used to regulate general translation rates. Extracellular cues, such as insulin, activate a signalling cascade that triggers 4E-BP hyperphosphorylation and release from eIF4E 19 , In addition to the phosphorylation-mediated changes that regulate global translation, proteolytic cleavage of translation factors can inhibit cellular protein synthesis.
For example, the apoptotic protein caspase-3 cleaves eIF4G and PABP 21 , 22 , and cleavage of these factors by viral proteases serves as a common and successful mechanism to interfere with the translation of cellular mRNAs Thoma and colleagues, unpublished results. The association of the 43S ribosomal complex with an mRNA is targeted not only by regulators of global translation, but also by RNA-binding proteins that modulate the translation of specific mRNAs. Here we discuss three different mechanisms by which RNA-binding proteins achieve this goal.
Steric blockage. The iron regulatory proteins IRP 1 and 2 control iron homeostasis, in part, by regulating the translation of the ferritin heavy- and light-chain mRNAs, which encode the two subunits of this iron storage protein. Translational repression is ineffective when the IRE is moved to a more distal position from the cap, presumably because this manipulation provides sufficient space in the cap-proximal region for binding of the 43S complex 26 , This mechanism seems to operate by steric hindrance, because replacing the IRE—IRP interaction by an RNA-binding interaction that involves other proteins with no physiological function in eukaryotic translation — such as the spliceosomal protein U1A with its corresponding RNA-binding sequence — can fully recapitulate translational repression Interfering with the eIF4F complex.
The cytoplasmic-polyadenylation-element-binding protein CPEB regulates the translation of maternal mRNA during vertebrate oocyte maturation and early development.
Other regulators have also been found to function as message-specific 4E-BPs. During anteroposterior axis formation in the early Drosophila melanogaster embryo, the mRNA that encodes the posterior determinant Nanos becomes concentrated — and is specifically translated — at the posterior pole of the D.
It is noteworthy that neither Maskin nor Cup were shown to directly prevent the recruitment of the 43S pre-initiation complex. Cap-independent inhibition of early initiation steps. Translation inhibition by IRP and message-specific 4E-BPs target steps of the translation-initiation pathway that are mediated by the cap structure. A recent example describes a regulator that inhibits the stable association of the 43S ribosomal complex with mRNA in a cap-independent manner.
Although both the cap structure and the poly A tail contribute to the translation of msl-2 mRNA, regulation occurs independently of either of these structures 36 , Translational repression by Sxl affects the stable association of the small ribosomal subunit with the mRNA, because the formation of 48S complexes is inhibited in the presence of Sxl. Taken together, these data indicate the interesting possibility that the repressor complex that is assembled around Sxl on msl-2 mRNA arrests the scanning 43S pre-initation complex.
Other mechanisms. This factor has been identified as the ribosomal protein L13a Curiously, dissociation of L13a from ribosomes after phosphorylation does not seem to affect global ribosome function Although the precise translational step that is affected by phosphorylated L13a has not been determined, repressed Cp mRNA is not found in association with polysomes, and translation inhibition requires the poly A tail as well as eIF4G and PABP 39 , These results implicate L13a in the regulation of translation initiation.
Translation can also be controlled at late-initiation and post-initiation steps. Translational repression of LOX is independent of the poly A tail and also occurs when translation is driven in a cap-independent manner by the encephalomyocarditis virus EMCV or the classical swine fever virus CSVF IRESs, which indicates that this type of regulation targets a late step in initiation 43 , So, these regulators seem to prevent the binding of the 60S ribosomal subunit to the 40S subunit at the initiation codon 44 Fig.
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