Stages of protein synthesis

The mechanismsof protein synthesis in prokaryotes and eukaryotes differ slightly in detail, but the prokaryotic mechanism is used as a general model  

Early steps in protein synthesis in prokaryotes formation of the 30S preinitiation complex and 70S initiation complex. 

30 kcal = 7.5 x 4). Recall that the synthesis of eaeh molecule of ammoacyl tRNA consumes two high-energy phosphate bonds (ATP AMP -I- PPi PPi + 2Pi). (In these ealeulations, the one GTP moleeule expended in the formation of the initiation eomplex is not included beeause of its small eontribution to the overall synthesis of a polypeptide.) 

Refilling of the A site. After transloeation has occurred, the A site is again available for a charged tRNA molecule with a correct anticodon. When this occurs, the series of elongation reactions is repeated. 

Termination. When a chain termination codon is reached, no aminoaeyl tRNA is available that can fill the A site, so chain elongation stops. Since the polypeptide chain is still attached to the tRNA oeeupying the P site, release of the protein is 

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FIGURE 6.1 The different stages of protein synthesis. Transcription and processing of RNA messages occur within the nucleus. The mRNA is then transported into the cytoplasm for translation and post-translational modifications. 

These three developments soon led to recognition of the major stages of protein synthesis and ultimately to the elucidation of the genetic code that specifies each amino acid. 

TABLE 27-5 Components Required for the Five Major Stages of Protein Synthesis in E. coli... 

Having looked at the structures of ribosomes and tRNAs, we now consider in detail the five stages of protein synthesis. 

During the first stage of protein synthesis, taking place in the cytosol, aminoacyl-tRNA synthetases esterify the 20 amino acids to their corresponding tRNAs. Each enzyme is specific for one amino acid and one or more corresponding tRNAs. Most organisms have one aminoacyl-tRNA synthetase for each amino acid. For amino acids with two or more corresponding tRNAs, the same enzyme usually aminoacylates all of them. 

The third stage of protein synthesis is elongation. Again, our initial focus is on bacterial cells. Elongation requires (1) the initiation complex described above, (2) aminoacyl-tRNAs, (3) a set of three soluble cytosolic proteins called elongation factors (EF-Tu, EF-Ts, and EF-G in bacteria), and (4) GTP. Cells use three steps to add each amino acid residue, and the steps are repeated as many times as there are residues to be added. 

In allosteric enzymes, the activity of the enzyme is modulated by a non-covalently bound metabolite at a site on a protein other than the catalytic site. Normally, this results in a conformational change, which makes the catalytic site inactive or less active. Covalent modulated enzymes are interconverted between active and inactive forms by the action of other enzymes, some of which are modulated by allosteric-type control. Both of these control mechanisms are responsive to changes in cell conditions and typically the response time in allosteric control is a matter of seconds as compared with minutes in covalent modulation. A third type of control, the control of enzyme synthesis at the transcription stage of protein synthesis (see Appendix 5.6), can take several hours to take effect. 

Oxazolidinones such as linezolid act at the early stage of protein synthesis, preventing the formation of the initiation complex between the 30S subunit, mRNA and fmet-tRNA. 

In this section, we describe the three basic stages of protein synthesis initiation, elongation, and termination. These three processes are fairly similar between prokaryotes and eukaryotes, with the two exceptions being that more protein factors have been identified as necessary for eukaryotic protein synthesis, and that transcription and translation are physically linked in prokaryotes but not in eukaryotes. Note that the reactions will be schematized as a single ribosome transversing the mRNA, but as shown in Figure 26.3, translation actually occurs on polyribosomes. 

Three key steps in the elongation stage of protein synthesis are required for the addition of each amino acid. Because these steps are repeated for each peptide bond formed, this is sometimes called the elongation cycle. The central theme in elongation is that the fully assembled ribosomal complex functions as a ribonucleoprotein machine which rapidly moves 50 to 30 down the mRNA, much like a ratchet. At the center of this complex are two binding sites which line up over a pair of triplet codons, as shown in Figure 26.11. These two sites are called the P site, for peptidyl (or polypeptide), and the A site, for aminoacyl (or acceptor). A third site, called the E site for tRNA exit site, is also a functional component of the ribosome, but for reasons of clarity, it is not included in the figures. 

The first stage of protein synthesis is initiation. Proteins called initiation factors are required to mediate the formation of a translation complex composed of an mRNA molecule, the small and large ribosomal subunits, and the initiator tRNA. This initiator tRNA recognizes the codon AUG and carries the amino acid methionine. 

Recall Prepare a flow chart showing the stages of protein synthesis. 

Outline the elongation stage of protein synthesis and describe the roles of the elongation factors (EEs) and GTP in the process. Locate the aminoacyl-tRNAs and peptidyl-tRNAs in the A or P sites of the ribosome during one cycle of elongation. 

At the present time there is no apparent explanation for the inhibition of the later stage of protein synthesis by wheat gluten, but this analysis suggests that the differences in response to these three dietary proteins involves more differences in amino acid composition and identifies the portion of the pathway which should be the subject of further research. 

Trichothecenes bind to eukaryotic ribosomes and inhibit protein synthesis (Pestka, Zhou, Moon, Chung, 2004). Different trichothecenes interfere with initiation, elongation and termination stages of protein synthesis. They are also immunosuppressive. Acute trichothecene mycotoxicosis are rare, but when ingested in high doses by farm animals they cause nausea, vomiting and diarrhea. DON is also called a vomitoxin or food refusal factor (Bennett Klich, 2003). Trichothecenes are not classifiable as to their carcinogenicity to humans (Class 3) (lARC). 

In experiments with cell-free rat-liver preparations, thioproline (L-thiazo-lidine-4-carboxylic acid Fig. 2) specifically inhibits the incorporation of -proline into ribosomal-bound nascent protein. S-thioproline is incorporated by a pathway similar to proline, since poly-C carrying the coding triplet for proline (CCQ enhances this incorporation. The isolation of S-thioproline-tiansfer RNA is a strong argument in favour of a competition mechanism with proline, at an early stage of protein synthesis. 

Polycistronic forms of messenger RNA, carrying information for several proteins, as we have seen are typical of viruses and are found in bacteria during the study of synthesis of proteins coded in one operon. However, in this case also we are forced to ask whether this RNA functions in the stage of protein synthesis on polysomes as a single linear system or as a sum of independently working cistrons, producing different numbers of protein molecules. 

In contrast to its deoxy template, RNA exhibits a wider variety of interactions with proteins, because the three RNA species have a wider range of functions, involving various stages of protein synthesis. Table 2 lists some of the proteins we wish to consider. Proteins interacting with RNA include those that trim RNA before ribosome formation. Since in eukaryotes there are approximately 70 ribosomal structural proteins, it is expected, and realized that these structural proteins undergo multiple interactions with the species of RNA. For example, evidence suggests that the interaction of 5 S RNA with the L5, L18, and L25 proteins situated in the large ribosomal subunit is involved in translocation of the peptidyl tRNA. 

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