Termination of Polypeptide Synthesis

Stage 4 Termination of Polypeptide Synthesis Requires a Special Signal... 

Initiation factors contribute to the ribosome complex with the messenger RNA and the initiator methionyl-tRNA. Elongation factors assist the binding of all the other tRNAs and the translocation reaction that must occur after each peptide bond is made. Termination factors recognize a stop signal and lead to the termination of polypeptide synthesis and the release of the polypeptide chain and the messenger from the ribosome. 

The genetic code consists of nucleosides in groups of three that is, it is a triplet code. Only 61 triplets code for amino acids the remaining three code for the termination of polypeptide synthesis. 

The alpha chain of human hemoglobin has 141 amino acids in a single polypeptide chain. Calculate the minimum number of bases on DNA necessary to code for the alpha chain. Include in your calculation the bases necessary for specifying the termination of polypeptide synthesis. 

Only 61 triplets code for amino acids the remaining three code for termination of polypeptide synthesis. 

Throughout the ribosome cycle, dynamic protein-mRNA interactions are functionally important in the initiation, elongation, and termination of polypeptide synthesis. In addition, more stable associations between proteins and mRNAs have been observed, particularly in eukaryotic cells. These messenger ribonucleoprotein complexes (mRNPs) occur both in polyribosomes and free in the cytosol, some of the latter being either temporarily or permanently unavailable for translation. Thus, protein-mRNA interactions contribute to the efficiency with which mRNAs are translated. 

Figure 20.24 The physiological pathway of polypeptide synthesis. The flux-generating step is that catalysed by the aminoacyl-tRNA synthetases, indicated by the broad arrow. The assumption implicit in this interpretation is that the physiological pathway starts with the intracellular amino acids and ends with the peptide that is formed in the elongation and termination processes. For the majority of enzymes, the concentration of intracellular amino acids is higher than the K, for the synthetase (Chapter 3).

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. 

An understanding of protein synthesis, the most complex biosynthetic process, has been one of the greatest challenges in biochemistry. Eukaryotic protein synthesis involves more than 70 different ribosomal proteins 20 or more enzymes to activate the amino acid precursors a dozen or more auxiliary enzymes and other protein factors for the initiation, elongation, and termination of polypeptides perhaps 100 additional enzymes for the final processing of different proteins and 40 or more kinds of transfer and ribosomal RNAs. Overall, almost 300 different macromolecules cooperate to synthesize polypeptides. Many of these macromolecules are organized into the complex three-dimensional structure of the ribosome. 

Elongation continues until the ribosome adds the last amino acid coded by the mRNA. Termination, the fourth stage of polypeptide synthesis, is signaled by the... 

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. 

The elongation phase of polypeptide synthesis and its termination are described in Example 17.8. The A site is shown to be filled by AA2-tRNA where the codon xxx is located. 

The inventory of phylogenetically relevant components offered by ribosomes comprises no less than three RNA molecules and about fifty proteins. Aminoacyl-tRNA synthetases and protein factors assisting the initiation, elongation and termination reactions of polypeptide synthesis constitute further probes of potential usefulness to delineate the unfolding of the early lineages. 

Premature termination of mRNA synthesis is mediated through translation of the leader peptide. The two tryptophan codons make translation of the leader polypeptide... 

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. 

The majority of mitochondria-destined preproteins seem to be imported after completion of polypeptide synthesis (Pfanner et al., 1997 Herrmann and Neupert, 2000). Preproteins are targeted to the outer membrane, often via N-terminal signals, where they bind to translo-case components of the outer membrane (TOM). Preproteins subsequently translocate across the general import pore (Ryan and Pfanner, 1998). Extra complexity is introduced into matrix-imported preproteins because they must also translocate across the inner membrane (Fig. 2). This is achieved via interactions of the preproteins with the translocase... 

Termination codon, stop codon, punctuation codon a sequence of three nucleotides in mRNA, which signals the end of polypeptide synthesis and release of the polypeptide in the process of Protein biosynthesis (see). 5 -tlAA (see Ochre codon), 5 -UAG (see Amber codon) and UGA are T.c. 

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. 

In the Merrifield method, the C-terminal amino acid is joined as a benzyl ester to the solid polymer support and then the polypeptide chain is extended one amino acid at a time from the N-terminal end. The advantage of polypeptide synthesis on a solid support is that the polymer beads with the peptide chains anchored on them are completely insoluble in the solvents used in the synthesis. Furthermore, excess reagents (e.g., DCC) and by-products (e.g., DCU) are removed after each step simply by washing the polymer beads. When synthesis is completed, the polypeptide is released from the polymer beads by cleavage of the benzyl ester. The steps in solid-phase synthesis of a polypeptide are summarized in Figure 27.11. 

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