Which of the following is not found at the site of protein synthesis

Abstract

Protein biosynthesis. The clinical subject is defects in mitochondrial oxidative phosphorylation, deficiency in mitochondrial translation. Additional topics are as follows: protein synthesis in the mitochondria, mitochondrial encephalomyopathy with lactic acid and stroke-like episodes, protein synthesis directed by the nucleus, the ribosome, structure of transfer RNA (tRNA), initiation and amino acid tRNA synthase, elongation and peptidyltransferase ribozyme, termination, inhibitors of protein synthesis, proteins synthesized in the cytoplasm but destined for mitochondria, proteins destined for the nucleus, and proteins destined for other sites including the plasma membrane and secretion from the cell. The chapter ends with a summary, reading list, review multiple-choice questions, and case-based problem.

9 Differences Between Eukaryotic and Prokaryotic Protein Synthesis

The overall scheme of protein synthesis is similar in all living cells. However, there are significant differences between bacteria and eukaryotes. These are summarized in Table 13.04 and discussed in the following sections. Note that eukaryotic cells contain mitochondria and chloroplasts, which have their own DNA and their own ribosomes. The ribosomes of these organelles operate similarly to those of bacteria and will be considered separately below. In eukaryotic protein synthesis, it is usually the cytoplasmic ribosomes that translate nuclear genes. Several aspects of eukaryotic protein synthesis are more complex. The ribosomes of eukaryotic cells are larger and contain more rRNA and protein molecules than those of prokaryotes. In addition, eukaryotes have more initiation factors and a more complex initiation procedure.

Table 13.04. Comparison of Protein Synthesis

Eukaryotic ribosomes are larger and more complex than those of prokaryotes.

A few aspects of protein synthesis are actually less complex in eukaryotes. In prokaryotes, mRNA is polycistronic and may carry several genes that are translated to give several proteins. In eukaryotes, each mRNA is monocistronic and carries only a single gene, which is translated into a single protein. In prokaryotes, the genome and the ribosomes are both in the cytoplasm, whereas in eukaryotes the genome is in the nucleus. Consequently, coupled transcription and translation is not possible for eukaryotes (except for their organelles; discussed later).

Both prokaryotes and eukaryotes have a special initiator tRNA that recognizes the start codon and inserts methionine as the first amino acid. In prokaryotes, this first methionine has a formyl group on its amino group (i.e., it is N-formyl-methionine), but in eukaryotes unmodified methionine is used.

9.1 Initiation, Elongation, and Termination of Protein Synthesis in Eukaryotes

Initiation of protein synthesis differs significantly between prokaryotes and eukaryotes. Eukaryotic mRNA has no ribosome-binding site (RBS). Instead recognition and binding to the ribosome rely on a component that is lacking in prokaryotes: The cap structure at the 5′ end, which is added to eukaryotic mRNA before it leaves the nucleus (see Chapter 12: Processing of RNA). Cap-binding protein (one of the subunits of eIF4) binds to the cap of the mRNA.

Eukaryotes also have more initiation factors than prokaryotes and the order of assembly of the initiation complex is different (see Table 13.05). Two different complexes assemble before binding to mRNA. The first is the 43S pre-initiation complex. This is an assembly of the small 40S subunit of the ribosome attached to several eukaryotic initiation factors (eIFs). These include eIF1, eIF1A, eIF3, and eIF5. This binds the charged initiator tRNA, Met-tRNAiMet, plus eIF2. The second complex, the cap-binding complex, contains cap-binding protein (eIF4E), eIF4G, eIF4A, eIF4B, and poly(A)-binding protein (PABP).

Table 13.05. Translation Factors: Prokaryotes vs Eukaryotes

	Prokaryotes	Eukaryotes
Initiation	IF1	eIF1A
IF2	eIF5B (GTPase)
IF3	eIF1
	eIF2 (α, β, γ) (GTPase)
	eIF2B (α, β, γ, δ, ɛ)
	eIF3 (13 subunits)
	eIF4A (RNA helicase)
	eIF4B (activates eIF4A)
	eIF4E (cap-binding protein)
	eIF4G (eIF4 complex scaffold)
	eIF4H
	eIF5
	eIF6
	PABP (Poly(A)-binding protein)
Elongation	EF-Tu	eEF1A
EF-Ts	eEF1B (2–3 subunits)
	SBP2
EF-G	eEF2
Termination	RF1	eRF1
RF2
RF3	eRF3
Recycling	RRF
EF-G
	eIF3
	eIF3j
	eIF1A
	eIF1

Functionally homologous factors are in the same row.

Adapted from Table 1 of Rodnina MV and Wintermeyer W. (2009) Recent mechanistic insights into eukaryotic ribosomes. Curr. Op. Cell Biol. 21: 435–443.

Eukaryotic mRNA is recognized by its cap structure (not by base pairing to rRNA).

During eukaryotic initiation, cap-binding complex first attaches to the mRNA via its cap. Next, the poly(A) tail is bound by PABP so that the mRNA forms a ring. This structure can now bind the 43S assembly. In order to align the Met-tRNAiMet with the correct AUG codon, the two structures work together to scan each codon from the 5′ end. This scanning process uses energy from ATP (Fig. 13.29). Normally, the first AUG is used as the start codon (see Box 13.02 for exceptions), although the sequence surrounding the AUG is important. The consensus is GCCRCCAUGG (R=A or G). If its surrounding sequence is too far from consensus an AUG may be skipped. Once a suitable AUG has been located, eIF5 joins the complex, which in turn allows the 60S subunit to join and the cap-binding protein, eIF2, eIF1, eIF3, and maybe eIF5 to depart. eIF5 uses energy from GTP to accomplish this remodeling of the ribosome.

Figure 13.29. Assembly of the Eukaryotic Initiation Complex

(A) The cap-binding complex includes poly(A)-binding protein (PABP), eIF4A, eIF4B, eIF4E, and eIF4G, which is in an unphosphorylated state when unbound to mRNA. ATP transfers phosphates to the complex to make it competent for binding the mRNA. (B) The 43S initiation complex forms bringing the small ribosomal subunit together with the tRNAimet. This complex uses GTP to attach the tRNA to the 40S subunit via eIF2. In addition, initiation factors eIF1, eIF1A, eIF3, eIF5, and eIF2B guide and make the complex competent to bind to the 5′-UTR of mRNA. (C) The mRNA is recognized by the cap-binding complex via the connections between eIF4E and PABP which bind the 5′ and 3′ ends of the mRNA, respectively. These two connections cause the rest of the mRNA to loop out. When this is established, then the 43S pre-initiation complex can attach and start scanning for the first AUG. After pausing at the first AUG, then the 50S subunit of the ribosome can bind and initiate translation.

Box 13.02

Internal Ribosome Entry Sites

Although most eukaryotic mRNA is scanned by the 40S subunit to find the first AUG, exceptions do occur. Sequences known as internal ribosome entry sites (IRES) are found in a few mRNA molecules. As the name indicates, these allow ribosomes to initiate translation internally, rather than at the 5′ end of the mRNA. IRES sequences were first found in certain viruses that have polycistronic mRNA despite infecting eukaryotic cells. In this case, the presence of IRES sequences in front of each coding sequence allows a single mRNA to be translated to give multiple proteins. The best known examples are members of the Picornavirus family, which includes poliovirus (causative agent of polio) and rhinovirus (one of the agents of common cold).

More recently, it has been found that a few special mRNA molecules encoded by eukaryotic cells themselves also possess IRES sequences. During major stress situations, such as heat shock or energy deficit, synthesis of the majority of proteins is greatly decreased. Much of this regulation occurs at the initiation stage of translation (discussed later). However, a few proteins are exempted from this down-regulation as they are needed under stress conditions. The mRNAs encoding these proteins often contain an IRES sequence. In these cases, the mRNA carries only a single coding sequence and the IRES is located in the 5′-UTR, between the 5′ end of the mRNA and the start of the coding sequence. This allows translation to be initiated at the IRES even in the absence of the standard initiation/scanning procedure.

The next stage is elongation (Fig. 13.30). Of all the stages of translation, elongation in bacteria and eukaryotes is the most similar. As in bacteria, elongation factors work to decode the mRNA and bind the tRNA into the A-site of the ribosome. Rather than EF-Tu and EF-Ts, eukaryotes use eEF1A to deliver the tRNA using GTP hydrolysis for energy and eEF1B to replace the depleted GDP with fresh GTP. The only difference is that eukaryotic elongation factors include more subunits. The remaining steps are the same. The peptidyl transferase activity of the 28S rRNA of the large subunit links the incoming amino acid to the polypeptide chain. Then elongation factor eEF2 (direct counterpart to bacterial EF-G) uses GTP to drive the conformational changes in the ribosome and ratchet the tRNAs from the P- and A-sites into the E- and P-sites. Elongation continues until a stop codon enters the A-site.

Figure 13.30. Beginning Eukaryotic Translation Elongation

Once the eukaryotic 40S subunit complex finds the first AUG, then the remaining 60S subunit and associated factors combine to form the final 80S ribosome.

Eukaryotic termination differs from prokaryotic termination in two ways. First, rather than having two different release factors (RF1 and RF2) to recognize different stop codons, eukaryotes have a single release factor (eRF1) that recognizes all three stop codons. eRF1 binds the stop codon, but this does not affect peptide bond formation. Instead, eRF3 carrying a GTP molecule binds to eRF1. GTP hydrolysis then rearranges the factors and the final amino acid attaches to the polypeptide. Therefore, eukaryotes require GTP for polypeptide completion, whereas in bacteria, RF1 or RF2 is sufficient.

Finally, as in bacteria, eukaryotic ribosomes are recycled. eIF3 triggers the release of the 60S subunit, and then eIF1 releases the final tRNA. An additional factor, eIF3j, then removes the mRNA. The components are then recycled.

Protein Synthesis

Protein synthesis represents the major route of disposal of amino acids. Amino acids are activated by binding to specific molecules of transfer RNA and assembled by ribosomes into a sequence that has been specified by messenger RNA, which in turn has been transcribed from the DNA template. Peptide bonds are then formed between adjacent amino acids. Once the polypeptide chain has been completed the subsequent folding, post-translational amino acid modifications and protein packaging are all determined by the primary sequence of amino acids. The rate of protein synthesis is controlled by the rate of transcription of specific genes, by the number and state of aggregation of ribosomes and by modulation of the rate of initiation of peptide synthesis.

A Mechanism of Action of Inhibitors of Protein Synthesis

Protein synthesis occurs in the cytoplasm on ribonucleoprotein particles, the ribosomes. Messenger RNA, which contains within its nucleotide sequence the code to direct the synthesis of one or several polypeptide chains, is synthesized by RNA polymerase on the DNA template and is transported into the cytoplasm, where it becomes bound to the ribosomes and directs the placement of amino acyl-transfer RNAs in the proper sequence. An amino acid, which has been activated and esterified to a specific species of tRNA, is bound to the ribosomal acceptor site by virtue of codon–anticodon interactions. Peptidyl transferase, an integral part of the ribosome, catalyzes the formation of a peptide bond between the carboxyl group of the nascent peptide (bound as peptidyl-tRNA to the ribosomal donor site) and the amino group of the new amino acid. The resultant peptidyl tRNA is translocated to the donor site by a GTP-requiring enzyme, freeing the acceptor site for the attachment of the next amino acyl-tRNA (Watson, 1970).

The relationship between protein synthesis and the physiological expression of radiation damage has been explored primarily with the use of inhibitors of protein synthesis. The conclusions drawn from these studies are based on two assumptions: first, that the inhibition affects one and only one biochemical reaction, and second, that this specific biochemical reaction has no rapid indirect effects on the general metabolism of the cell.

Puromycin, which functions as an analog of amino acyl-tRNA (Morris and Schweet, 1961; Rabinovitz and Fisher, 1962), appears to inhibit protein synthesis in prokaryotic and eukaryotic cells by releasing incomplete polypeptide chains from the ribosome (Allen and Zamecnik, 1962; Nathans, 1964). Cycloheximide can inhibit the initiation, elongation, or termination of protein synthesis in eukaryotic cells by blocking translocation, thereby preventing further movement of the ribosome along the messenger RNA (Obrig et al., 1971; Rajalakshmi et al., 1971). Chloramphenicol inhibits the synthesis of protein in bacteria and selectively inhibits protein synthesis in the mitochondria and chloroplasts of the eukaryotic cells that have been studied (Sager, 1972). This antibiotic binds to the large ribosomal subunit (Vazquez, 1965) and interferes with peptide bond formation (e.g., Traut and Monro, 1964). Streptomycin specifically inhibits microbial and mitochondrial protein synthesis by binding to the small ribosomal subunit (Davies, 1964; Cox et al., 1964) and causing misreading of the genetic code (Davies et al., 1964).

16.3 Cell Proliferation Provides Critical Context for Interpreting Protein Turnover: A Key Proteodynamic Mechanism

Protein synthesis plays a chief role in ensuring proteome fidelity [15]. With advancing age, misfolded, damaged, and aggregated proteins accumulate across tissues [14,16–19]. Protein damage triggers mechanisms to reestablish proteostasis [20], with protein turnover a key mechanism for removing and replacing damaged proteins (see the top panel of Fig. 16.1). Some, but not all, evidence suggests that aging decreases bulk protein synthesis rates [21] and mechanisms of protein degradation [22,23]. It follows, then, that this age-related decrease of protein turnover exacerbates accumulation of damaged proteins, which presents further challenges to proteostatic maintenance and ultimately contributes to cellular, organ, and organism dysfunction [23,24].

Robust analytical approaches are important for understanding changes in protein turnover with aging and to assess the efficacy of interventions to maintain or increase protein turnover. The most commonly used measurement of protein content is insufficient because both increases and decreases in protein turnover can occur without a change in protein content [25,26]. Therefore, methods are needed that capture dynamic processes. Many have used stable isotopically labeled amino acids to provide a relatively acute measurement of the dynamics of protein synthesis. As detailed in other publications [27], results gleaned from labeled amino acid measurements must still be interpreted with their limitations in mind. Specifically, the acute labeling period used in these study designs can bias the measured protein synthesis rates toward rapidly turning over and abundant proteins, without accounting for the proteins that are less abundant and those that turn over more slowly.

Measuring protein synthesis rates over longer periods of time (days to weeks) using the stable isotope of water, deuterium oxide (D2O), provides a robust approach that is feasible in vitro as well as in free-living animals and humans. Making measurements over prolonged periods and in free-living environments facilitates collection of data that reflect differences that accumulate over time, and overcomes the bias toward proteins that are more abundant and turn over more rapidly. One advantage of using D2O for protein synthesis measurements is the ability to simultaneously measure the incorporation of deuterium into new DNA, which provides a sensitive method to measure DNA replication [28]. Simultaneously measuring protein and DNA synthesis rates provides mechanistic insight into protein synthetic responses [29]. To explain, when cells proliferate, protein mass is doubled in the growth phases so that two daughter cells have the full complement of genetic material and protein machinery [30]. In tissues such as skeletal muscle, where the predominant cell type is postmitotic skeletal muscle fibers, growth is accompanied by DNA synthesis in supportive cells. Donation of nuclear material from supportive cells to the postmitotic cells [31] minimizes changes to the cytosolic volume to DNA ratio [32]. In these scenarios, both protein and DNA synthesis rates would increase. By comparison, when the cells synthesize new proteins to replace damaged and degraded proteins, rates of DNA synthesis are not concomitantly increased. Therefore, during growth/proliferation the ratio of protein synthesis rates to DNA synthesis rates essentially stays the same, whereas with proteostatic maintenance or tissue remodeling, the ratio of protein synthesis to DNA synthesis increases (Fig. 16.1, bottom panel). Being able to make these simultaneous measurements has clarified the paradoxical observation that both aged and long-lived models appear to have slow rates of protein turnover. In the next section, we discuss what these measurements tell us about activation of proteostatic mechanisms during interventions that involve energetic stress signaling or growth restriction.

7.14.6.2.4 Fungal protein biosynthesis inhibitors

Protein synthesis has long been considered as an attractive target in the development of antimicrobial agents, in light of the widespread use of antibacterial antibiotics that target the specific areas of this process. However, application of this idea to the field of antifungal therapy is not an easy task, due to the eukaryotic rather than prokaryotic nature of fungi, and therefore the great degree of similarity between the fungal and mammalian protein synthesis machineries. Two soluble elongation factors show some fungal specificity: EF3, a factor that is required by fungal ribosomes only, and EF2, which has been demonstrated to possess at least one functional distinction from its mammalian counterpart. The sordarins are the most important family of antifungal agents acting at the protein synthesis level. Compounds in this class inhibit in vitro translation in C. albicans, C. tropicalis, C. kefyr, and C. neoformans, to varying degrees.117 The lack of activity of the sordarins against C. krusei, C. glabrata, and C. parapsilosis, in comparison with their extremely high levels of potency against C. albicans, suggests that these compounds have a highly specific binding site, which may also be the basis for the greater selectivity of these compounds in inhibiting fungal, but not the mammalian, protein synthesis. The most advanced inhibitors of fungal protein biosynthesis are analogs of the natural product lead sordarin lead, GR135402 (see Figure 13).146,147 Its spectrum of activity includes C. albicans, C. tropicalis, and C. neoformans, where impressive antifungal activity has been observed in vitro (MIC<1 μg mL−1 in many cases), but not in other Candida or Aspergillus species, which severely restricts its potential as a quality lead. Efficacy has been seen in animal models,147 although at high dose and predominantly via nonoral routes, reflecting the potential for rapid clearance and limited oral bioavailability.

tRNA Synthetases

Protein biosynthesis at the ribosome results in the conversion of nucleic acid genetic information into the polypeptides essential for cellular function. Peptide bond formation requires numerous protein and nucleotide cofactors in addition to the ribosome itself, and is tightly regulated to ensure accurate translation. Transfer RNA (tRNA) synthetases are key players in this synthetic pathway. Each enzyme in the family attaches a specific amino acid to its corresponding tRNA, thus defining the rules of the genetic code. Organisms use up to 20 tRNA synthetases to generate aminoacyl-tRNAs, one for each of the 20 standard amino acids. While all tRNA synthetases carry out the same fundamental chemistry, they are distinguished by their structure, regulation, and often by novel functions outside their canonical role in protein biosynthesis.

3.3 Mechanisms of translational control

Protein synthesis is controlled by the efficiency of the translational apparatus, which is determined by the factors that influence translation initiation (Kaufman, 1994). Initiation of translation involves consecutive recruitment of the small and large subunits of ribosomes to specific mRNAs, with the formation of an active ribosome at the initiation site. The predominant mechanism for control of protein synthesis appears to be reversible phosphorylation, under the control of selected protein kinases and protein phosphatases. The targets of covalent modification in this case are translational components, specifically the initiation and elongation factors (Hershey, 1991). The eukaryotic Initiation Factor 2 alpha (eIF-2α), which promotes the binding of initiator tRNA to the 40S ribosomal subunit, is an example of a factor that is regulated in this manner. Phosphorylation of eIF-2α is correlated with inhibition of protein synthesis in a range of eukaryotes (Rhoads, 1993) and our recent studies with L. littorea concur (Larade and Storey, 2002a). In L. littorea hepatopancreas, the total content of eIF-2α was constant in three groups: aerobic control snails, snails exposed to 24 h under a N2 gas atmosphere, and snails given 1 h aerated recovery after 24 h anoxia exposure. However, in response to anoxia exposure, the content of phosphorylated eIF-2α rose ~ 15-fold compared with aerobic controls (Fig. 3.3) (Larade and Storey, 2002a). This was reversed rapidly during aerobic recovery with phosphoprotein content reduced again to control levels within 1 h post-anoxia. These data support the concept that anoxia exposure in L. littorea, and likely also in other anoxia-tolerant molluscs, stimulates a substantial suppression of protein synthesis, a proposal that is further supported by the direct measurements of the protein biosynthesis rates (discussed earlier) and by the changes in the distribution of ribosomes between polysomes versus monosomes (discussed below).

Introductory Comments

Protein synthesis in mitochondria follows the same basic steps seen in bacterial and eukaryotic cytoplasmic translational systems. The process is divided into four major stages – initiation, elongation, termination and ribosome recycling. Given the presumed prokaryotic origin of mitochondria, it is expected that the process of protein synthesis in this organelle will be more closely related to that of bacteria than to that of the eukaryotic cell cytoplasm. This idea is borne out by studies of the translational machinery; however, there are a number of interesting and fundamental differences between mitochondrial and bacterial translation. Further, there are clearly distinct differences in how this process takes place in the mitochondria of different organisms. The most detailed studies have been carried out with the mammalian and yeast mitochondrial systems as summarized below.

Low nutritional intake and low protein intake

Muscle protein synthesis rate is reported to be reduced 30% in the elderly, but there is controversy as to the extent to which this reduction is due to nutrition, disease, or physical inactivity rather than aging.82,83 It is recognized by some that protein intake in elders should exceed the 0.8 g/kg per day recommend intake.84 Muscle protein synthesis is also decreased in fasting elderly subjects, especially in specific muscle fractions like mitochondrial proteins,85 and thus, the anorexia of aging and its underlying mechanisms contribute to sarcopenia by reducing protein intake.

Muscle protein synthesis is directly stimulated by amino acid and essential amino acids intake,86 and protein supplementation has been explored in the prevention of sarcopenia. However, many interventional studies have not reported a significant increase muscle mass or protein synthesis with a high protein diet even when accompanied by resistance training.87–89 The lack of effect of protein intake on protein synthesis stimulation may have several explanations.38 A higher splanchnic extraction of dietary amino acids has been already reported.90 This could limit the delivery of dietary amino acids to the peripheral skeletal muscle.