Which two cell structures work together in the process of protein synthesis?

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

ProkaryotesEukaryotesInitiationIF1eIF1AIF2eIF5B (GTPase)IF3eIF1eIF2 (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)eIF4HeIF5eIF6PABP (Poly(A)-binding protein)ElongationEF-TueEF1AEF-TseEF1B (2–3 subunits)SBP2EF-GeEF2TerminationRF1eRF1RF2RF3eRF3RecyclingRRFEF-GeIF3eIF3jeIF1AeIF1

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.

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.

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.

Abstract

Protein synthesis is the process of synthesizing new, or the regeneration of existing, functional peptides. This process is highly regulated, involving a network of upstream and downstream factors that modulate mRNA translation initiation and elongation through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 signaling can be upregulated or inhibited in response to a variety of stimuli that dictate protein balance. This chapter will review the process of protein synthesis, with a focus on the intracellular regulation of mTORC1 signaling in muscle. The regulation of myogenesis, that is, the process of developing new and regeneration of existing muscle cells, will also be detailed in the context of intramuscular regulation of protein synthesis. This chapter will provide the basis for understanding the protein synthetic responses to endogenous and exogenous modulators of human health and function, such as exercise, nutrition, disease, and aging.

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).

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.

1 Theory

Protein synthesis is a highly regulated process that is controlled by a complex network of proteins. Many of these proteins are essential for viability and mutations are not well tolerated, often affecting the fidelity or rates of protein synthesis that can dramatically affect growth (Carr-Schmid et al., 1999; Hinnebusch, 1985). Several forms of cellular stress can also trigger the repression of protein synthesis. Moreover, many bacterial and viral pathogens target the host translation machinery (Gradi et al., 1998; Honjo et al., 1968; Shenton et al., 2006). For these reasons, it is essential to have quantitative methods for measuring protein synthesis in vivo under a variety of conditions. One commonly used method is the incorporation of [35 S]-methionine into total cellular proteins during an interval of time that allows protein synthesis to be measured. This method has the advantages of being performed in vivo and without modification of the yeast strains to be studied unless the strain is auxotrophic for methionine biosysnthesis. It cannot, however distinguish between the inhibition of translation at different stages of translation (initiation, elongation or termination). This requires polyribosomal analysis to be performed in addition to [35 S]-methionine incorporation.

3.9 Roles of Protein Molecules in Heat Tolerance

Ubiquitination serves as a versatile posttranslational modification that mediates growth and development of all eukaryotic species. Ubiquitin is a stable, highly conserved, and universally expressed protein. The covalent attachment of ubiquitin to a lysine residue of select proteins can regulate stability, activity, and trafficking. Genome sequencing has revealed the extent to which plants rely on protein ubiquitination to regulate organismal processes. For example, over 6% of A. thaliana protein-coding genes are dedicated to the ubiquitin 26S proteasome system (UPS) (Vierstra, 2009). In plant species, the UPS regulates fundamental processes such as embryogenesis, photomorphogenesis, and organ development (Thomann et al., 2005; Sonoda et al., 2009; Pokhilko et al., 2011). In addition to regulating these fundamental processes, the UPS has recently emerged as a major player in plant responses to abiotic stresses.

The UPS functions within the cytoplasm and nucleus to modulate the levels of regulatory proteins and to remove misfolded or damaged proteins that may accumulate as a result of exposure to abiotic stress. One of the first indications that the UPS was involved in regulating plant stress tolerance was the observation that expression of polyubiquitin genes is stress-regulated (Christensen et al., 1992; Genschik et al., 1992; Sun and Callis, 1997). Ubiquitin is encoded by multiple polyubiquitin genes (UBQ3, UBQ4, UBQIO, UBQ11, and UBQ14) that contain three to six ubiquitin-coding regions in tandem (Callis et al., 1995). Following translation, nascent polyubiquitin proteins are proteolytically processed into ubiquitin monomers (Vierstra, 1996). The pool of free ubiquitin molecules is regulated through differential expression of the polyubiquitin genes (Christensen et al., 1992; Genschik et al., 1992; Sun and Callis, 1997). Specifically, transcript abundance of Arabidopsis UBQ14 is increased during heat stress (Sun and Callis, 1997). Similarly, high temperatures also induce the expression of multiple polyubiquitin genes in tobacco, potato, and maize (Christensen et al., 1992; Garbarino et al., 1992; Genschik et al., 1992). In fact, overexpression of a single mono-ubiquitin gene enhances tolerance to multiple stresses without adversely affecting growth and development under favorable conditions (Guo et al., 2008). Transgenic tobacco overexpressing a wheat polyubiquitin gene, containing a single ubiquitin repeat, were more tolerant of cold, high salinity, and drought conditions compared with control plants. The stress-induced expression of polyubiquitin genes is consistent with the role of the UPS in turning over damaged proteins to mitigate the negative effects of environmental stress (Lyzenga and Stone, 2012). Defects in 26S proteasome function also alter plant tolerance to various environmental stresses. The 26S proteasome is an ATP-dependent protease complex consisting of a proteolytic 20S complex capped on one or both ends by a 19S regulatory particle. Access to the active sites of the 20S complex is regulated by the regulatory particle that mediates substrate recruiting, unfolding, translocation into the proteolytic chamber of the 20S, and recycling of ubiquitin molecules (Strickland et al., 2000; Navon and Goldberg, 2001).

Protein synthesis elongation factor Tu (EF-Tu) is a protein that plays a central role in the elongation phase of protein synthesis in bacteria and organelles including mitochondria and plastids in plants. The cytosolic homolog of EF-Tu in plants is EF-1α. The polypeptide elongation cycle proceeds in three steps:

EF-Tu binds GTP and aminoacyl-tRNA, which leads to the codon-dependent placement of this aminoacyl-tRNA at the A site of the ribosome, GTP hydrolysis, and release of EF-Tu-GDP from the ribosome;