Termination of protein synthesis

Fig. 8.4 Outline of the main events in protein synthesis initiation, elongation, translocation and termination. AUG is an initiation codon on the mRNA it codes for Af-fomiylmelhionine and initiates the formation of the 70S rihosome. UAG is a termination codon it does not code for any amino acid and brings about termination of protein synthesis.

Potter, M. D., and Nicchitta, C. V. (2002). Endoplasmic reticulum-bound ribosomes reside in stable association with the translocon following termination of protein synthesis. J. Biol. Chem. 277, 23314-23320. 

Figure 10 Alteration of the genetic code for incorporation of non-natural amino acids, (a) In nonsense suppression, the stop codon UAG is decoded by a non-natural tRNA with the anticodon CUA. In vivo decoding of the UAG codon by this tRNA is in competition with termination of protein synthesis by release factor 1 (RFl). Purified in vitro translation systems allow omission of RF1 from the reaction mixture, (b) A new codon-anticodon pair can be created using four-base codons such as GGGU. Crystal structures of these codon-anticodon complexes in the ribosomal decoding center revealed that the C in the third anticodon position interacts with both the third and fourth codon position (purple line) while the extra A in the anticodon loop does not contact the codon.(c) Non-natural base pairs also allow creation of new codon-anticodon pairs. Shown here is the interaction of the base Y with either base X or (hydrogen bonds are indicated by red dashes).

Deactivation of an RF is feasible, however, in prokaryotes. In E. coli, three RFs are involved in the termination process, and there is a degree of codon specificity for two of them. RFl recognizes the UAG and UAA stop codons, while RF2 recognizes the UGA and UAA stop codons [16, 42]. RFS, which is not codon specific, stimulates the activity of the other two. Thus, if RF 1 is deactivated, UAA and UGA remain functional termination codons, but UAG should no longer signal for termination of protein synthesis. To test this hypothesis, the Chamberlin lab investigated a mutant strain of E. coli that produces a faulty RFl [29]. 

Klaholz BP, Myasnikov AG, Van Heel M (2004) Visualization of release factor 3 on the ribosome during termination of protein synthesis. Nature 427 862-865... 

The ribosome can carry two aminoacyl-tRNAs simultaneously. In the chain elongation stage, the growing polypeptide is carried on one of these tRNAs. The chain is transferred to the second tRNA, which adds its amino acid to the growing peptide, and displaces the first tRNA. The ribosome then moves one codon along the mRNA to allow the next to be read. Termination of protein synthesis involves the release of the completed polypeptide, expulsion of the last tRNA, and dissociation of the ribosome from the mRNA. This is signaled by specific termination codons (UAA, UAG, or UGA) in the mRNA and requires the participation of various release factors. 

FIGURE 27-26 Termination of protein synthesis in bacteria. Termination occurs in response to a termination codon in the A site. First, a release factor, RF (RF-1 or RF-2, depending on which termination codon is present), binds to the A site. This leads to hydrolysis of the ester linkage between the nascent polypeptide and the tRNA in the P site and release of the completed polypeptide. Finally, the mRNA, de-acylated tRNA, and release factor leave the ribosome, and the ribosome dissociates into its 30S and 50S subunits. 

Codons are composed of three nucleotide bases usually presented in Ihe mRNA language of A, G, C, and U. They are always written 5 —>3. Of the 64 possible three-base combinations, 61 code for the twenty common amino acids and three signal termination of protein synthesis (translation). Altering the nucleotide sequence in a codon can cause sient mutations (the altered codon also codes for the original amino acid), missense mutations (the altered codon codes for a different amino acid), or nonsense mutations (the altered codon is a termination... 

Requirements include all the amino acids that eventually appear in the finished protein, at least one specific type of tRNA for each amino acid, one aminoacyl-tRNA synthetase for each amino acid, the mRNA coding for the protein to be synthesized, fully competent ribosomes, protein factors needed for initiation, elongation, and termination of protein synthesis, and ATP and GTP as energy sources. 

The two possible consequences of base-pair substitution are that the gene encodes for either no amino acid or the wrong amino acid. Effects can range from minor results to termination of protein synthesis. 

Four classes of LDL receptor mutations have been identified. Class 1 mutations are characterized by the failure of expression of the receptor protein. It is possible, however, that a modified protein is produced but it is not recognized as an LDL receptor protein. Class 2 mutations involve a nonsense mutation (premature termination of protein synthesis Chap. 17), and result in a defect in the transfer of the receptor from the endoplasmic reticulum to the cell membranes. This class of mutation is common in Afrikaners and Lebanese. The Watanabe heritable hyperlipidemic rabbit (WHHL) is an animal model which has a Class 2 defect and has been used extensively for the study of familial hypercholesterolemia. Class 3 mutations result in abnormal binding of LDL. This can be caused by alterations in the amino acid sequence of Domain 1. Class 4 mutations are those with defective internalization due to the receptor s inability to be located in coated pits. This is the result of mutations in the fifth, C-terminal domain. 

Unspecific permeability is prevented in the translationally active translocon because the tunnel is occupied by the peptide chain being polymerized. Similarly, the pore is blocked by BiP proteins, prominent intraluminal chaperones, after dissociation of the ribosome from the complex (53). In fact, it has been argued that the BiP locks form a smaller barrier for uncharged polar molecules than for charged ones. Furthermore, the dissociation of ribosomes from translocon complexes is delayed after the termination of protein synthesis. When a nontranslating ribosome is associated with the translocon complex, they form a transitional low-selectivity channel between the cytosol and the ER lumen (5). 

Although rRNA is paramount in the process of translation, protein factors also are required for the efficient synthesis of a protein. Protein factors participate in the initiation, elongation, and termination of protein synthesis. P-loop NTPases of the G-protein family play particularly important roles. Recall that these proteins serve as molecular switches as they cycle between a GTP-bound form and a GDP-bound form (Section 15.1.2). 

Termination of protein synthesis occurs when the polypeptidyl chain is transferred to a water molecule rather than an aminoacyl-tRNA. What molecule provides energy for this transfer ... 

The mechanism for termination of protein synthesis in eukaryotes requires... 

Translation is the process whereby the mRNA sequence directs the amino acid sequence during protein synthesis. Twenty-one amino acids are involved in protein synthesis and each is specified by a three-nucleotide sequence known as a codon. Because there are 64 possible codons, most amino acids are specified by more than one codon. In addition, two codons do not code for amino acids but always signal termination of protein synthesis (stop codons), and one codon,... 

In summary, the codon TGA, which normally codes for termination of protein synthesis is programmed from a distance to encode the 21st amino acid selenocysteine a special tRNA is loaded in a unique and unorthodox way, incorporating a selenocysteine residue which is synthesised de novo at the tRNA level by special enzymes highly specialised proteins are required to recognise and bind the secondary mRNA structures and the tRNA specialised elongation factors have to compete with canonical ones and with release factors. No wonder that chemists and biochemists ask what are the unique properties of selenocysteine compared to cysteine (Amer, 2010) to justify the involvement of so many molecular partners (Allmang, Wurth, Krol, 2009). 

Figure 30.25 Termination of protein synthesis. A release factor recognizes a stop codon m the A site and stimulates the release of the completed protein from the tRNA in the P site.

The cycle of peptide-chain elongation continues until one of the three stop codons (UAA, UAG, UGA) is reached. There is no aminoacyl-tRNA complementary to these codons, and instead a termination factor or a release factor (RF) with bound GTP binds to the ribosome and induces hydrolysis of both the aminoacyl-linkage and GTP, thereby releasing the completed polypeptide chain from the ribosome. The 475 amino acid-long sequence of rabbit liver RF has been deduced from its cDNA sequence, and it shows 90% homology with mammalian trypto-phanyl-tRNA synthetase (Lee et al., 1990). It has also been reported that for efficient and accurate termination, an additional fourth nucleotide (most commonly an A or a G) after the stop codon is required (Tate and Brown, 1992). The exact role of the fourth nucleotide in the termination of protein synthesis is not fully understood at present. 

Poole. E.. and W. Tate. 2000. Release factors and their role as decoding proteins specificity and fidelity for termination of protein synthesis. Biochim. Biophys. Acta 1493 1-11. 

A stop signal is required for the termination of protein synthesis. The codons UAA, UAG, and UGA are the stop signals. These codons are not recognized by any tRNAs, but they are recognized by proteins called release factors (Figure 12.15). One of two protein release factors (RF-1 or RF-2) is required, as is GTP, which is bound to a third release factor, RF-3. RF-1 binds to UAA and UAG, and RF-2 binds to UAA and UGA. RF-3 does not bind to any codon, but it does facilitate the activity of the other two release factors. Either RF-1 or RF-2 is bound near the A site of the ribosome when one of the termination codons is reached. The release factor not only blocks the binding of a new aminoacyl-tRNA but also affects the activity of the peptidyl transferase so that the bond... 

Recall Describe the role of stop codons in the termination of protein synthesis. 

Amber codon, nonsense codon the sequence UAG in mRNA. It does not code for any of the 20 proteogenic amino acids, and it results in the termination of protein synthesis (premature termination when UAG is produced by mutation of a sense codon). Potential precursors for the production of UAG by mutation are UCG (serine), UAU and UAC (tyrosine) and CAG (glutamine). 

Regulatory mechanisms at the level of mRNA translation could also lead to gross metabolic changes. The mechanism of protein synthesis has been exhaustively studied [5], and many components have been implicated. Changes in each of these components—ribosomes, factors involved in the ribosomal binding of mRNA, in the initiation and termination of protein synthesis, and in polypeptide chain elongation, tRNA, and the components responsible for its acylation and subsequent transfer to the polysomal complex—could potentially lead to alteration in the rate, extent, or fidelity of protein synthesis. 

The hydrolysis of GTP is coupled to the operation of many of the factors that control initiation, elongation and termination of protein synthesis in order to provide directionality and specificity to these processes [45]. The free energy of hydrolysis of ATP is utilized indirectly to provide specificity in proofreading processes, such as the cleavage of incorrectly activated amino acids on aminoacyl-tRNA or aminoacyl-AMP molecules [46]. GTP hydrolysis in the reaction cycle initiated by the... 

FIGURE 11 The cascade of regulated alternative splicing events controlling Sxl, transformer, and double-sex ex-pression in Drosophila melanogaster. The positions of translational stop codons that will cause premature termination of protein synthesis are indicated by Stop. See text for further details. 

Codons have now been determined for all 20 amino acids. A total of 64 codons is possible from the triplet combinations of A, G, C, and U (see Table 18.11). Three of these, UGA, UAA, and UAG, are stop signals that code for the termination of protein synthesis. All the other codons specify amino acids one amino acid can have several codons. For example, glycine has four codons GGU, GGC, GGA, and GGG. The triplet AUG has two roles in protein synthesis. At the beginning of an mRNA, the codon AUG signals the start of protein synthesis. In the middle of a series of codons, AUG codes for the amino acid methionine. 

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