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| Mechanism of Protein Synthesis |
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| The events in protein synthesis are better known in bacteria than in eukaryotes. Although these are thought to be similar in the two groups there are some differences. The following description refers mainly to protein synthesis in bacteria on the 70S ribosome. |
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| Protein synthesis is a highly complex and an elaborate process and involves the following steps. |
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| It is the step in which each of the participating amino acid reacts with ATP to form amino acid AMP complex and pyrophosphate. The reaction is catalyzed by a specific amino acid activating enzyme called aminoacyl-tRNA synthetase in the presence of Mg2+. There is a separate aminoacyl tRNA synthetase enzyme for each kind of amino acid. Much of the energy released by the separation of phosphate groups from ATP is trapped in the amino acid AMP complex. The complex remains temporarily associated with the enzyme. The amino acid AMP enzyme complex is called an activated amino acid. The pyrophosphate is hydrolyzed to two in organic phosphates (2pi), |
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Amino acid + ATP
 Amino acid AMP enzyme complex + ppi |
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| fig. 22.5 Activation of Amino Acids |
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| It is the step in which the amino acid AMP-enzyme complex joins with the amino acid binding site of its specific tRNA, where its COOH group bonds with the OH group of the terminal base triplet CCA. The reaction is catalyzed by the same enzyme, aminoacyl tRNA synthetase. The resulting tRNA-amino acid complex is called a charged tRNA. AMP and enzyme are released. The released enzyme can activate and attach another amino acid molecule to another tRNA molecule. The energy released by change of ATP to AMP is retained in the amino acid-tRNA complex. This energy is later used to drive the formation of peptide bond when amino acids link together and form a polypeptide. |
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| Amino acid AMP Enzyme complex + tRNA |
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| The tRNA amino acid complex moves to the ribosomes, the site of protein synthesis. |
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| It is the step in which the smaller and the larger subunits of ribosome are joined together. This is brought about by mRNA chain. The latter joins the smaller ribosomal subunit with the help of the first codon by a base pairing with an appropriate sequence on rRNA. The combination of the two is called initiation complex. The larger subunit later joins the small subunit, forming active ribosome. Activation of ribosome by mRNA requires proper concentration of Mg++. |
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| It is the step in which the amino acids are assembled into a polypeptide chain. It involves 3 events: initiation, elongation and termination of polypeptide chain. |
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| Initiation of Polypeptide Chain |
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| The mRNA chain has at its 5 end an “initiator” or “start” codon (AUG or GUG) that signals the beginning of polypeptide formation. This codon lies close to the P site of the ribosome. The amino acid formylmethionine (methionine in eukaryotes) initiates the process. It is carried by tRNA having an anticodon UAC which bonds with the initiator codon AUG of mRNA. Initiation factors (IF1, IF2 and IF3) and GTP promote the initiation process. |
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| The large ribosomal subunit now joins the small subunit to complete the ribosome. At this stage, GTP is hydrolysed to GDP. The ribosome has formylmethionine bearing tRNA at the P site. Later, the formylmethionine is changed to normal methionine by the enzyme deformylase in prokaryotes. If not required, methionine is later separated from the polypeptide chain by a proteolytic enzyme aminopeptidase. |
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| Elongation of Polypeptide Chain |
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| fig. 22.6 Elongation of Polypeptide Chain on a Ribosome |
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| The above figure shows, |
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| A. A charged tRNA arriving at the A site, reading its codon on the mRNA. |
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| B. Amino acid of tRNA at P site is ready to be transferred to the amino acid of tRNA at A site. |
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| C. Amino acids are joined by peptide bond and tRNA is discharged from P site. |
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| D. Peptide chain-carrying tRNA is translocated to P site, making A site free to receive another charged tRNA. |
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| Three elongation factors (EF Tu, EF Ts and EF G) assist in the elongation of the polypeptide chain. A charged tRNA molecule along with its amino acid, proline, for example, enters the ribosome at the A site. Its anticodon GGA locates and binds with the complementary codon CCU of mRNA chain by hydrogen bonds. The amino acid methionine is transferred from its tRNA onto the newly arrived proline tRNA complex where the two amino acids join by a peptide bond. The process is catalyzed by the enzyme peptidyl transferase located on the ribosome. In this process, the linkage between the first amino acid and its tRNA is broken, and the -COOH group now forms a peptide bond with the free -NH2 group of the second amino acid. Thus, the second tRNA carries a dipeptide, formylmethionineproline. The energy required for the formation of a peptide bond comes from the free energy released by separation of amino acid (formylmethionine or methionine) from its tRNA. |
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| The first tRNA, now uncharged, separates from mRNA chain at the P site of the ribosome and returns to the mixed pool of tRNAs in the cytoplasm. Here, it is now available to transport another molecule of its specific amino acid. |
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| Now the ribosome moves one codon along the mRNA in the 3 direction. With this, tRNAdipeptide complex at the A site is pulled to the P site. This process is called translocation. It requires GTP and a translocase protein called EF-G factor. The GTP is hydrolysed to GDP and inorganic phosphate to release energy for the process. |
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| At this stage, a third tRNA molecule with its own specific amino acid, arginine, for example arrives at the A site of the ribosome and binds with the help of anticodon AGA to the complementary codon UCU of the mRNA chain. The dipeptide formylmethionineproline is shifted from the preceding tRNA on the third tRNA where it joins the amino acid arginine again with the help of peptidyl transferase enzyme. The dipeptide, thus, becomes a tripeptide, formyl-methionine-proline-arginine. The second tRNA being now uncharged, leaves the mRNA chain, vacating the P site. The tRNAtripeptide complex is translocated from A site to P site. |
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| The entire process involving arrival of tRNA-amino acid complex, peptide bond formation and translocation is repeated. As the ribosome moves over the mRNA, all the codons of mRNA arrive at the A site one after another, and the peptide chain grows. Thus, the amino acids are linked up into a polypeptide in a sequence communicated by the DNA through the mRNA. A polypeptide chain which is in the process of synthesis is often called a nascent polypeptide. |
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| The growing polypeptide chain always remains attached to its original ribosome, and is not transferred from one ribosome to another. Only one polypeptide chain can be synthesized at a time on a given ribosome. |
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| Termination and Release of Polypeptide Chain |
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| At the terminal end of mRNA chain there is a stop, or terminator codon (UAA, UAG or UGA). It is not joined by the anticodon of any tRNA amino acid complex. Hence, there can be no further addition of amino acids to the polypeptide chain. The linkage between the last tRNA and the polypeptide chain is broken by three release factors. (RF 1, RF 2 and RF 3) and GTP. The release is catalyzed by the peptidyl transferase enzyme, the same enzyme that forms the peptide bonds. The ribosome jumps off the mRNA chain at the stop codon and dissociates into its two subunits. The completed polypeptide (amino acid chain) becomes free in the cytoplasm. |
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| The ribosomes and the tRNAs on release from the mRNA can function again in the same manner and result in the formation of another polypeptide of the same protein. |
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| The just released polypeptide is a straight, linear exhibiting a primary molecule, structure. It may lose some amino acids from the end with the help of a peptidase enzyme, and then coil and fold on itself to acquire secondary and tertiary structure. It may even combine with other polypeptides, to have quaternary structure. |
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| The proteins synthesized on free polysomes are released into the cytoplasm and function as structural and enzymatic proteins. The proteins formed on the polysomes attached to ER pass into the ER channels and are exported as cell secretions by exocytosis after packaging in the Golgi apparatus. |
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| When the ribosome has moved sufficiently down the mRNA chain towards 3 end, another ribosome takes up position at the initiator codon of mRNA, and starts synthesis of a second molecule of the same polypeptide chain. At any given time, the mRNA chain will, therefore, carry many ribosomes over which are similar polypeptide chains of varying length, shortest near the initiator codon and longest near the terminator codon. A row of ribosomes joined to the mRNA molecule, is called a polyribosome, or a polysome. Synthesis of many molecules of the same polypeptide simultaneously from one mRNA molecule by a polysome is called translational amplification. |
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| fig. 22.7 Summary of the Events in Protein Synthesis |
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| One GTP is hydrolysed to GDP as each successive amino acid-tRNA complex attaches to the A site of the ribosome. A second GTP is broken down to GDP as the ribosome moves to each new codon in the mRNA. One ATP is hydrolysed to AMP during amino acid activation. Thus, the formation of each peptide bond uses 3 high-energy molecules, one ATP and two GTP. |
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| An interesting aspect of protein synthesis is that the DNA and ribosomes are located at different sites in the cell. |
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| Location of instruction centre (DNA) and manufacturing centre (ribosomes) at different sites in a cell is advantageous. If both were in the nucleus, the manufacturing centre would be far away from the energy sources and raw materials; and if both were in the cytoplasm, the information centre would be exposed to respiratory breakdown. The nuclear envelope preserves stability of the DNA by protecting it from respiratory destruction. The message in the DNA in the form of genes (codes) are, permanent, authentic master documents from which working copies are prepared in the form of mRNAs, as and when required by the cell. |
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