The general structural characteristics of ribosomes and their self-assembly process are discussed in Chapter 34. These particulate entities serve as the machinery on which the mRNA nucleotide sequence is translated into the sequence of amino acids of the specified protein. The translation of the mRNA commences near its 5′ end with the formation of the corresponding amino terminus of the protein molecule. The message is decoded from 5′ to 3′, concluding with the formation of the carboxyl terminus of the protein. Again, the concept of polarity is apparent. As described in Chapter 36, the transcription of a gene into the corresponding mRNA or its precursor first forms the 5′ end of the RNA molecule. In prokaryotes, this allows for the beginning of mRNA translation before the transcription of the gene is completed. In eukaryotic organisms, the process of transcription is a nuclear one, while mRNA translation occurs in the cytoplasm, precluding simultaneous transcription and translation in eukaryotic organisms and enabling the processing necessary to generate mature mRNA from the primary transcript.
Initiation Involves Several Protein–RNA Complexes
Initiation of protein synthesis requires that an mRNA molecule be selected for translation by a ribosome (Figure 37–6). Once the mRNA binds to the ribosome, the ribosome must locate the initiation codon thereby setting the correct reading frame on the mRNA, and translation begins. This process involves tRNA, rRNA, mRNA, and at least 10 eukaryotic initiation factors (eIFs), some of which have multiple (three to eight) subunits. Also involved are GTP, ATP, and amino acids. Initiation can be divided into four steps: (1) dissociation of the ribosome into its 40S and 60S subunits; (2) binding of a ternary complex consisting of the initiator methionyl-tRNA, (met-tRNAi), GTP, and eIF-2 to the 40S ribosome to form the 43S preinitiation complex; (3) binding of mRNA to the 40S preinitiation complex to form the 48S initiation complex; and (4) combination of the 48S initiation complex with the 60S ribosomal subunit to form the 80S initiation complex.
Diagrammatic representation of the initiation phase of protein synthesis on an eukaryotic mRNA. Eukaryotic mRNAs contain a 5′ 7meG-cap (Cap) and 3′ poly(A) terminal [(A) n] as shown. Translation preinitiation complex formation proceeds in several steps: (1) activation of mRNA (right); (2) formation of the ternary complex consisting of met-tRNAi, initiation factor eIF-2, and GTP (left); (3) scanning in the 43S complex to locate the AUG initiator coding, forming the 48S initiation complex (center); and (4) formation of the active 80S initiation complex (bottom, center). (See text for details.) (GTP, •; GDP,°.) The various initiation factors appear in abbreviated form as circles or squares, for example, eIF-3, (
), eIF-4F, (4F), (
). 4•F is a complex consisting of 4E and 4A bound to 4G (see Figure 37–7
). The poly A binding protein, which interacts with the mRNA 3′-poly A tail, is abbreviated PAB. This collection of protein factors and the 40S ribosomal subunit comprise the 43S preinitiation complex, which after binding to mRNA, forms the 48S preinitiation complex.
Two initiation factors, eIF-3 and eIF-1A, bind to the newly dissociated 40S ribosomal subunit. This delays its reassociation with the 60S subunit and allows other translation initiation factors to associate with the 40S subunit.
Formation of the 43S Preinitiation Complex
The first step in this process involves the binding of GTP by eIF-2. This binary complex then binds to met tRNAi, a tRNA specifically involved in binding to the initiation codon AUG. (It is important to note that there are two tRNAs for methionine. One specifies methionine for the initiator codon, the other for internal methionines. Each has a unique nucleotide sequence; both are aminoacylated by the same methionyl-tRNA synthetase.) The GTP-eIF-2-tRNAi ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex, which is subsequently stabilized by association with eIF-3 and eIF-1A.
eIF-2 is one of two control points for protein synthesis initiation in eukaryotic cells. eIF-2 consists of α, β, and γ subunits. eIF-2α is phosphorylated (on serine 51) by at least four different protein kinases (HCR, PKR, PERK, and GCN2) that are activated when a cell is under stress and when the energy expenditure required for protein synthesis would be deleterious. Such conditions include amino acid or glucose starvation, virus infection, intracellular presence of large quantities of misfolded proteins, serum deprivation, hyperosmolality, and heat shock. PKR is particularly interesting in this regard. This kinase is activated by viruses and provides a host defense mechanism that decreases protein synthesis, including viral protein synthesis, thereby inhibiting viral replication. Phosphorylated eIF-2α binds tightly to and inactivates the GTP–GDP recycling protein eIF-2B. Thus preventing formation of the 43S preinitiation complex and blocking protein synthesis.
Formation of the 48S Initiation Complex
As described in Chapter 36 the 5′ termini of mRNA molecules in eukaryotic cells are “capped.” The 7meG-cap facilitates the binding of mRNA to the 43S preinitiation complex. A cap-binding protein complex, eIF-4F (4F), which consists of eIF-4E (4E) and the eIF-4G (4G)-eIF-4A (4A) complex, binds to the cap through the 4E protein. Then eIF-4B (4B) binds and reduces the complex secondary structure of the 5′ end of the mRNA through its ATP-dependent helicase activity. The association of mRNA with the 43S preinitiation complex to form the 48S initiation complex requires ATP hydrolysis. eIF-3 is a key protein because it binds with high affinity to the 4G component of 4F, and it links this complex to the 40S ribosomal subunit. Following association of the 43S preinitiation complex with the mRNA cap, and reduction (“melting”) of the secondary structure near the 5′ end of the mRNA through the action of the 4B helicase and ATP, the complex translocates 5′ → 3′ and scans the mRNA for a suitable initiation codon. Generally, this is the 5′-most AUG, but the precise initiation codon is determined by so-called Kozak consensus sequences that surround the AUG initiation codon:
Most preferred is the presence of a purine (Pu) at positions −3 and a G at position +4.
Role of the Poly(A) Tail in Initiation
Biochemical and genetic experiments in yeast have revealed that the 3′ poly(A) tail and the poly A binding protein, PAB1, are both required for efficient initiation of protein synthesis. Further studies showed that the poly(A) tail stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions. PAB (Figure 37–7) bound to the poly(A) tail, interacts with eIF-4G, and 4E subunits of cap-bound eIF-4F to form a circular structure that helps direct the 40S ribosomal subunit to the 5′ end of the mRNA and also likely stabilizes mRNAs from exonucleolytic degradation. This helps explain how the cap and poly(A) tail structures have a synergistic effect on protein synthesis. Indeed, differential protein-protein interactions between general and specific mRNA translational repressors and eIF-4E result in m7G Cap-dependent translation control (Figure 37–8).
Schematic illustrating the circularization of mRNA through protein-protein interactions between 7meG cap-bound elF4F and poly A tail-bound poly A binding protein. elF4F, composed of elF4A, 4E, and 4G subunits binds the mRNA 5′-7meG “Cap” (7meGpppX-) upstream of the translation initiation codon (AUG) with high affinity. The elF4G subunit of the complex also binds poly A binding protein (PAB) with high affinity. Since PAB is bound tightly to the mRNA 3′-poly A tail (5′-(X)nA(A)n AAAAAAAOH 3′), circularization results. Shown are multiple 80S ribosomes that are in the process of translating the circularized mRNA into protein (black curlicues), forming a polysome. Upon encountering a termination codon (here UAA), translation termination occurs leading to release of the newly translated protein and dissociation of the 80S ribosome into 60S, 40S subunits. Dissociated ribosomal subunits can recycle through another round of translation (see Figure 37–6).
Activation of eIF-4E by insulin and formation of the cap binding eIF-4F complex. The 4F-cap mRNA complex is depicted as in Figures 37–6 and 37–7. The 4F complex consists of eIF-4E (4E), eIF-4A, and eIF-4G. 4E is inactive when bound by one of a family of binding proteins (4E-BPs). Insulin and mitogenic growth polypeptides, or growth factors (eg, IGF-1, PDGF, interleukin-2, and angiotensin II) activate the PI3 kinase/AKT kinase pathways, which activate the mTOR kinase, and results in the phosphorylation of 4E-BP (see Figure 42–8). Phosphorylated 4E-BP dissociates from 4E, and the latter is then able to form the 4F complex and bind to the mRNA cap. These growth polypeptides also induce phosphorylation of 4G itself by the mTOR and MAP kinase pathways. Phosphorylated 4F binds much more avidly to the cap than does nonphosphorylated 4F, which stimulates 48S initiation complex formation and hence translation.
Formation of the 80S Initiation Complex
The binding of the 60S ribosomal subunit to the 48S initiation complex involves hydrolysis of the GTP bound to eIF-2 by eIF-5. This reaction results in release of the initiation factors bound to the 48S initiation complex (these factors then are recycled) and the rapid association of the 40S and 60S subunits to form the 80S ribosome. At this point, the met-tRNAi is on the P site of the ribosome, ready for the elongation cycle to commence.
The Regulation of eIF-4E Controls the Rate of Initiation
The 4F complex is particularly important in controlling the rate of protein translation. As described above, 4F is a complex consisting of 4E, which binds to the m7G cap structure at the 5′ end of the mRNA, and 4G, which serves as a scaffolding protein. In addition to binding 4E, 4G binds to eIF-3, which links the complex to the 40S ribosomal subunit. It also binds 4A and 4B, the ATPase-helicase complex that helps unwind the RNA (Figure 37–8).
4E is responsible for recognition of the mRNA cap structure, a rate-limiting step in translation. This process is further regulated by phosphorylation. Insulin and mitogenic growth factors result in the phosphorylation of 4E on Ser209 (or Thr210). Phosphorylated 4E binds to the cap much more avidly than does the nonphosphorylated form, thus enhancing the rate of initiation. Components of the MAP kinase, PI3K, mTOR, RAS, and S6 kinases pathways (see Figure 42–8) can all, under appropriate conditions, be involved in these regulatory phosphorylation reactions.
The activity of 4E is modulated in a second way, and this also involves phosphorylation; a set of proteins bind to and inactivate 4E. These proteins include 4E-BP1 (BP1, also known as PHAS-1) and the closely related proteins 4E-BP2 and 4E-BP3. BP1 binds with high affinity to 4E. The [4E]•[BP1] association prevents 4E from binding to 4G (to form 4F). Since this interaction is essential for the binding of 4F to the ribosomal 40S subunit and for correctly positioning it on the capped mRNA, BP-1 effectively inhibits translation initiation.
Insulin and other growth factors result in the phosphorylation of BP-1 at seven unique sites. Phosphorylation of BP-1 results in its dissociation from 4E, and it cannot rebind until critical sites are dephosphorylated. These effects on the activation of 4E explain in part how insulin causes a marked post-transcriptional increase of protein synthesis in liver, adipose, and muscle tissue.
Elongation Is Also a Multistep, Accessory Factor-Facilitated Process
Elongation is a cyclic process on the ribosome in which one amino acid at a time is added to the nascent peptide chain (Figure 37–9). The peptide sequence is determined by the order of the codons in the mRNA. Elongation involves several steps catalyzed by proteins called elongation factors (EFs). These steps are (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, (3) translocation of the ribosome on the mRNA, and (4) expulsion of the deacylated tRNA from the P- and E-sites.
Binding of Aminoacyl-tRNA to the A Site
In the complete 80S ribosome formed during the process of initiation, both the A site (aminoacyl or acceptor site) and E site (deacylated tRNA exit site) are free. The binding of the appropriate aminoacyl-tRNA in the A site requires proper codon recognition. Elongation factor 1A (EF1A) forms a ternary complex with GTP and the entering aminoacyl-tRNA (Figure 37–9). This complex then allows the correct aminoacyl-tRNA to enter the A site with the release of EF1A•GDP and phosphate. GTP hydrolysis is catalyzed by an active site on the ribosome; hydrolysis induces a conformational change in the ribosome concomitantly increasing affinity for the tRNA. As shown in Figure 37–9, EF1A-GDP then recycles to EF1A-GTP with the aid of other soluble protein factors and GTP.
Diagrammatic representation of the peptide elongation process of protein synthesis. The small circles labeled n − 1, n, n + 1, etc, represent the amino acid residues of the newly formed protein molecule (in N-terminal to C-terminal orientation) and the corresponding codons in the mRNA. EFIA and EF2 represent elongation factors 1 and 2, respectively. The peptidyl-tRNA, aminoacyl-tRNA, and exit sites on the ribosome are represented by P site, A site, and E site, respectively.
The α-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by the initiator met-tRNAi. This reaction is catalyzed by a peptidyl transferase, a component of the 28S RNA of the 60S ribosomal subunit. This is another example of ribozyme activity and indicates an important—and previously unsuspected—direct role for RNA in protein synthesis (Table 37–3). Because the amino acid on the aminoacyl-tRNA is already “activated,” no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site.
TABLE 37–3Evidence That rRNA Is a Peptidyl Transferase ||Download (.pdf) TABLE 37–3 Evidence That rRNA Is a Peptidyl Transferase
|• Ribosomes can make peptide bonds (albeit inefficiently) even when proteins are removed or inactivated. |
|• Certain parts of the rRNA sequence are highly conserved in all species. |
|• These conserved regions are on the surface of the RNA molecule. |
|• RNA can be catalytic in many other chemical reactions. |
|• Mutations that result in antibiotic resistance at the level of protein synthesis are more often found in rRNA than in the protein components of the ribosome. |
|• X-ray crystal structure of large subunit bound to tRNAs suggests detailed mechanism. |
The now deacylated tRNA is attached by its anticodon to the P site at one end and by the open CCA tail to the E site on the large ribosomal subunit (middle portion of Figure 37–9). At this point, elongation factor 2 (EF2) binds to and displaces the peptidyl tRNA from the A site to the P site. In turn, the deacylated tRNA is on the E site, from which it leaves the ribosome. The EF2-GTP complex is hydrolyzed to EF2-GDP, effectively moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of amino acid tRNA–EF1A-GTP and another cycle of elongation.
The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed peptidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second; prokaryotic ribosomes incorporate as many as 18 per second. Thus, the energy requiring process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached.
Termination Occurs When a Stop Codon Is Recognized
In comparison to initiation and elongation, termination is a relatively simple process (Figure 37–10). After multiple cycles of elongation culminating in polymerization of the specific amino acids into a protein molecule, the stop or terminating codon of mRNA (UAA, UAG, UGA) appears in the A site. Normally, there is no tRNA with an anticodon capable of recognizing such a termination signal. Releasing factor RF1 recognizes that a stop codon resides in the A site (Figure 37–10). RF1 is bound by a complex consisting of releasing factor RF3 with bound GTP. This complex, with the peptidyl transferase, promotes hydrolysis of the bond between the peptide and the tRNA occupying the P site. Thus, a water molecule rather than an amino acid is added. This hydrolysis releases the protein and the tRNA from the P site. Upon hydrolysis and release, the 80S ribosome dissociates into its 40S and 60S subunits, which are then recycled (Figure 37–7). Therefore, the releasing factors are proteins that hydrolyze the peptidyl-tRNA bond when a stop codon occupies the A site. The mRNA is then released from the ribosome, which dissociates into its component 40S and 60S subunits, and another cycle can be repeated.
Diagrammatic representation of the termination process of protein synthesis. The peptidyl-tRNA, aminoacyl-tRNA and exit sites are indicated as P site, A site, and E site, respectively. The termination (stop) codon is indicated by the three vertical bars and stop. Releasing factor RF1 binds to the stop codon in the A site. Releasing factor RF3, with bound GTP, binds to RF1. Hydrolysis of the peptidyl-tRNA complex is shown by the entry of H2O. N and C indicate the amino- and carboxy-terminal amino acids of the nascent polypeptide chain, respectively, and illustrate the polarity of protein synthesis. Termination results in release of the mRNA, the newly synthesized protein, free tRNA, 40S and 60S subunits, as well as RF1, GDP-bound RF3 and inorganic Pi, as shown at bottom right.
The P body is a cytoplasmic organelle involved in mRNA metabolism. Shown is a photomicrograph of two mammalian cells in which a single distinct protein constituent of the P body has been visualized using the cognate specific fluorescently labeled antibody. P bodies appear as small red circles of varying size throughout the cytoplasm. The cell plasma membranes are indicated by a solid white line, nuclei by a dashed line. Nuclei were counterstained using a fluorescent dye with different fluorescence excitation/emission spectra from the labeled antibody used to identify P bodies; the nuclear stain intercalates between the DNA base pairs and appears as blue/green. Modified from http://www.mcb.arizona.edu/parker/WHAT/what.htm. (Used with permission of Dr Roy Parker.)
Polysomes Are Assemblies of Ribosomes
Many ribosomes can translate the same mRNA molecule simultaneously. Because of their relatively large size, the ribosome particles cannot attach to an mRNA any closer than 35 nucleotides apart. Multiple ribosomes on the same mRNA molecule form a polyribosome, or “polysome” (Figure 37–7). In an unrestricted system, the number of ribosomes attached to an mRNA (and thus the size of polyribosomes) correlates positively with the length of the mRNA molecule.
Polyribosomes actively synthesizing proteins can exist as free particles in the cellular cytoplasm or may be attached to sheets of membranous cytoplasmic material referred to as endoplasmic reticulum. Attachment of the particulate polyribosomes to the endoplasmic reticulum is responsible for its “rough” appearance as seen by electron microscopy. The proteins synthesized by the attached polyribosomes are extruded into the cisternal space between the sheets of rough endoplasmic reticulum and are exported from there. Some of the protein products of the rough endoplasmic reticulum are packaged by the Golgi apparatus for eventual export (see Chapter 46). The polyribosomal particles free in the cytosol are responsible for the synthesis of proteins required for intracellular functions.
Nontranslating mRNAs Can Form Ribonucleoprotein Particles That Accumulate in Cytoplasmic Organelles Termed P Bodies
mRNAs, bound by specific packaging proteins and exported from the nucleus as ribonucleoproteins particles (mRNPs) sometimes do not immediately associate with ribosomes to be translated. Instead, specific mRNAs can associate with the protein constituents that form P bodies, small dense compartments that incorporate mRNAs as mRNPs (Figure 37–11). These cytoplasmic organelles are related to similar small mRNA-containing granules found in neurons and certain maternal cells. P bodies are sites of translation repression and mRNA decay. Over 35 distinct proteins have been suggested to reside exclusively or extensively within P bodies. These proteins range from mRNA decapping enzymes, RNA helicases and RNA exonucleases (5′-3′ and 3′-5′), to components involved in miRNA function and mRNA quality control. However, incorporation of an mRNP is not an unequivocal mRNA “death sentence.” Indeed, though the mechanisms are not yet fully understood, certain mRNAs appear to be temporarily stored in P bodies and then retrieved and utilized for protein translation. This suggests that an equilibrium exists where the cytoplasmic functions of mRNA (translation and degradation) are controlled by the dynamic interaction of mRNA with polysomes and P bodies.
The Machinery of Protein Synthesis Can Respond to Environmental Threats
Ferritin, an iron-binding protein, prevents ionized iron (Fe2+) from reaching toxic levels within cells. Elemental iron stimulates ferritin synthesis by causing the release of a cytoplasmic protein that binds to a specific region in the 5′ nontranslated region of ferritin mRNA. Disruption of this protein-mRNA interaction activates ferritin mRNA and results in its translation. This mechanism provides for rapid control of the synthesis of a protein that sequesters Fe2+, a potentially toxic molecule (see Figure 52–8). Similarly environmental stress and starvation inhibit the positive roles of mTOR (Figure 37–8; Figure 42–8) on promoting activation of eIF-4F and 48S complex formation.
Many Viruses Co-Opt the Host Cell Protein Synthesis Machinery
The protein synthesis machinery can also be modified in deleterious ways. Viruses replicate by using host cell processes, including those involved in protein synthesis. Some viral mRNAs are translated much more efficiently than those of the host cell (eg, encephalomyocarditis virus). Others, such as reovirus and vesicular stomatitis virus, replicate efficiently, and thus their very abundant mRNAs have a competitive advantage over host cell mRNAs for limited translation factors. Other viruses inhibit host cell protein synthesis by preventing the association of mRNA with the 40S ribosome.
Poliovirus and other picornaviruses gain a selective advantage by disrupting the function of the 4F complex. The mRNAs of these viruses do not have a cap structure to direct the binding of the 40S ribosomal subunit (see above). Instead, the 40S ribosomal subunit contacts an internal ribosomal entry site (IRES) in a reaction that requires 4G but not 4E. The virus gains a selective advantage by having a protease that attacks 4G and removes the amino terminal 4E binding site. Now the 4E–4G complex (4F) cannot form, so the 40S ribosomal subunit cannot be directed to host capped mRNAs, abolishing host cell protein synthesis. The 4G fragment can direct binding of the 40S ribosomal subunit to IRES-containing mRNAs, so viral mRNA translation is very efficient (Figure 37–12). These viruses also promote the dephosphorylation of BP1 (PHAS-1), thereby decreasing cap (4E)-dependent translation (Figure 37–8).
Picornaviruses disrupt the 4F complex. The 4E-4G complex (4F) directs the 40S ribosomal subunit to the typical capped mRNA (see text). However, 4G alone is sufficient for targeting the 40S subunit to the internal ribosomal entry site (IRES) of certain viral mRNAs. To gain selective advantage, some viruses (eg, poliovirus) express a protease that cleaves the 4E binding site from the amino terminal end of 4G. This truncated 4G can direct the 40S ribosomal subunit to mRNAs that have an IRES but not to those that have a cap (ie, host cell mRNAs). The widths of the arrows indicate the rate of translation initiation from the AUG codon in each example. Other viruses utilize distinct processes to effect selective initiation of translation on their cognate viral mRNAs via IRES elements.