Protein synthesis polypeptide elongation

The translation of the mRNA into proteins is the final step in the biological flow of information (see Fig. 6.1). Similar to other macromolecular polymerizations, protein synthesis can be divided into initiation, chain elongation, and termination. Critical players in this process are the aminoacyl transfer RNAs (tRNAs). These molecules form the interface between the mRNA and the growing polypeptide. Activation of tRNA involves the addition of an amino acid to its acceptor stem, a reaction catalyzed by an aminoacyl-tRNA synthetase. Each aminoacyl-tRNA synthetase is highly specific for one amino acid and its corresponding tRNA molecule. The anticodon loop of each aminoacyl-tRNA interacts... 

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. 

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5 -end to its 3 -end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 420). Each coding region has its own initiation codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA codes for only one polypeptide chain, that is, it is monocistronic. The process of translation is divided into three separate steps initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details. [Note Individual differences are mentioned in the text.]... 

The RNA genomes of single-stranded RNA bacterial viruses, such as Q/3, MS2, R17, and f2, are themselves mRNAs. Bacteriophage Q/3 codes for a polypeptide that combines with three host proteins to form an RNA-depen-dent RNA polymerase (replicase). The three host proteins are ribosomal protein SI and two elongation factors for protein synthesis EF-Tu and EF-Ts (see table 28.5). The Q/3 replicase functions exclusively with the Q/3 RNA plus strand template. It first makes a complementary RNA transcript (minus strand) and ultimately uses the minus strand as... 

We considered the structure of the ribosome in some detail in the previous chapter without referring to those sites that are functionally important in protein synthesis. Here we can localize some of the functional sites on the two ribosomal subunits (fig. 29.6). The mRNA binds to the smaller ribosomal subunit. The peptidyl transferase is an integral part of the 50S subunit, and the elongation factor EF-G binds to the 50S subunit. The nascent polypeptide chain exits through a channel in the 50S subunit. Two functional sites occur on... 

In perfused liver it also has effects on synthesis of most types of RNA and on amino acid uptake, and these may underlie much of the action on protein synthesis. For most effects on the liver there appears to be a delay of 15-60 min between the application of hormone and the first manifestation of the effect. Ribosomes from liver of hypophysectomized rats possess a lowered ability to carry out protein synthesis [86] and the defect can be reversed by administration of GH in vivo or in vitro. The effect may be at least partly on the rate of elongation of the growing polypeptide chain [85]. 

The chemical polymerization of even a moderately sized protein of a hundred amino acids in the laboratory is extremely laborious, and the yields of active product can often be low to zero (Kent and Parker, 1988). Cells accomplish this task by using an intricate mechanism which involves catalytic machinery composed of proteins, nucleic acids and their complexes, and synthesize polypeptide chains that are composed of hundreds of amino acids. This process is depicted in Fig. 2.4, and is described in the sections below. The basic components of the cellular protein synthesis apparatus, in all known biological systems, are ribosomes, which are aggregate structures containing over fifty distinct proteins, and three distinct molecules of nucleic acid known as ribosomal ribonucleic acid (ribosomal RNA or rRNA). The amino acids are brought to the ribosomes, the assembly bench , by an RNA molecule known appropriately as transfer RNA . Each of the twenty amino acids is specifically coupled to a set of transfer RNAs (discussed below) which catalyze their incorporation into appropriate locations in the linear sequence of polypeptide chains. Several other intracellular proteins known as init iation and elongation factors a re also required for protein synthesis. 

Vectorial translation [31,32]. Polypeptides are made on membrane-bound polysomes. Many of these proteins are synthesized with a 16-30 amino acid extension at the NH2-terminus. This signal sequence is hydrophobic in nature. Protein synthesis and translocation, into or across the membrane, are obligatorily linked. Therefore, the transmembrane movement is co-translational and it is coupled to the elongation of the polypeptide chain. Consequently, the completed polypeptide chain is never present in the compartment where it is synthesized. The polypeptides that do not yet cross the membrane are shorter than the mature protein. Addition of inhibitors of protein synthesis immediately arrest movement of the polypeptide across the membrane. 

The answer is c. (Murray, pp 452-467. Scriver, pp 3-45. Sack, pp 1-40. Wilson, pp 101-120.) In a general sense, the mechanism of protein synthesis in eukaryotic cells is similar to that found in prokaryotes however, there are significant differences. Cycloheximide inhibits elongation of proteins in eukaryotes, while erythromycin causes the same effect in prokaryotes. Thus, one is an antibiotic beneficial to humans, while the other is a poison. Cytoplasmic ribosomes of eukaryotes are larger, sedimenting at SOS instead of 70S. While eukaryotic cells utilize a specific tRNA for initiation, it is not formylated as in bacteria. Finally, eukaryotic mRNA always specifies only one polypeptide, as opposed to prokaryotic mRNA, which may specify the synthesis of more than one gene product per mRNA. 

One of the primary killers of children prior to immunization was upper respiratory tract infections by Corynebacterium diphtheriae. Toxin produced by a lysogenic phage that is carried by some strains of this bacteria causes the lethal effects. It is lethal in small amounts because it blocks protein synthesis. The viral toxin is composed of two parts. The B portion binds a cell s surface and injects the A portion into the cytosol of cells. The A portion ADP-ribosylates a histidine-derived residue of the elongation factor 2 (EF-2) known as diphthamide. This action completely blocks the ability of EF-2 to translocate the growing polypeptide chain. 

The second step in the elongation of protein synthesis is the peptidyl-transferase reaction in which a peptide bond is formed between the amino acid in the P site and the amino acid coupled to aminoacyl-tRNA in the A site. This reaction is facilitated by the intrinsic activity of the ribosome. As a result of this reaction, the growing polypeptide chain becomes elongated without moving forward. The movement or translocation of the dipeptide-tRNA from the A site to the P site is achieved by the action of EF-2, while another molecule of GTP is hydrolyzed. This process results in the relative movement of the mRNA by three nucleotides, so that a new codon becomes readable in the A site. The deacylated tRNA is pushed out of the ribosome after a transient halt at the so-called exit (E) site. At this point, all the components... 

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. 

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