Polypeptide chains, elongation protein synthesis

It is known that protein synthesis is necessary for BR-induced effects in root tissue (25), and BR-treatment increases nucleic acid and protein synthesis in bean stem (26). In the pea stem segment, kinetic studies with selected protein and nucleic acid synthesis inhibitors showed no evidence for competitive inhibition in polypeptide chain elongation, post-transcriptional polyA addition to heterogeneous RNA, DNA-dependent RNA synthesis, or of the DNA-dependent RNA polymerase... 

To this point, you have become familiar with the molecules that participate in protein synthesis and the genetic code, the language that directs the synthesis. We now investigate the actual process by which polypeptide chains are assembled. There are three major stages in protein synthesis initiation of the polypeptide chain, elongation of the chain, and termination of the completed polypeptide chain. 

Cells incubated in medium without one or all of the essential amino acids also show a decline in protein synthesis over the first hour, involving an inhibition of both polypeptide chain elongation and initiation (Van Venrooij et al., 1972 Vaughan et al, 1971). Inhibition is fully reversible on readdition of the amino acid(s), even in the presence of actinomycin D, and, again, initiation appears to be the more sensitive of the two steps. It is not known whether this effect is a result of some general control of the rate of initiation by the intracellular level of all 20 amino acids or their charged tRNA s, or whether it is an indirect result of a decrease in the level of ATP (Van Venrooij et al, 1972). 

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. 

Ballinger, D. G., and Pardue, M. L., 1983, The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide chain elongation rates. Cell 33 103. 

Initiation of protein synthesis in mammalian cells proceeds by a complex process whereby the assembly of mRNA, the ribosome, and initiator met-tRNAf into an initiation complex is catalyzed by a group of proteins called initiation factors. By definition, these proteins are not required for polypeptide chain elongation. A detailed discussion of this process, and the role of individual initiation factors can be found in this volume (Kaempfer, 1984), or in other recent reviews (Benne and Hershey, 1978 Jagus et al., 1981) which also contain pertinent references. Nine initiation factors have been highly purified from rabbit reticulocytes, and still other factors have been described which may serve auxiliary functions, or indeed may qualify as initiation factors in their own right. A very brief (and oversimplifed) description of the role of the nine, characterized initiation factors is listed below, along with the step in initiation complex formation in which each participates ... 

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. 

A large number of components are required for the synthesis of a polypeptide chain. These include all the amino acids that are found in the finished product, the mRNA to be translated, tRNAs, functional ribosomes, energy sources, and enzymes, as well as protein factors needed for initiation, elongation, and termination of the polypeptide chain. 

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.]... 

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. 

Several architectural paradigms are known for polyketide and fatty acid synthases. While the bacterial enzymes are composed of several monofunctional polypeptides which are used during each cycle of chain elongation, fatty acid and polyketide synthases in higher organisms are multifunctional proteins with an individual set of active sites dedicated to each cycle of condensation and ketoreduction. Peptide synthetases also exhibit a one-to-one correspondence between the enzyme sequence and the structure of the product. Together, these systems represent a unique mechanism for the synthesis of biopolymers in which the template and the catalyst are the same molecule. 

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. 

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. 

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