Index >> Biosynthesis of proteins >> Protein Synthesis on 70s Ribosomes

	Protein Synthesis on 70s Ribosomes The mechanism of protein synthesis on 70S ribosomes includes following stages: 1. The transcription The process of protein synthesis is started by the uncoiling of strands of DNA molecule. One strand of DNA molecule acts as a template for the formation of mRNA. In the presence of RNA polymerase enzyme the mRNA is formed according to the triplet codes of DNA by the copying or transcription process. As soon as the mRNA is formed it leaves the nucleus and reaches in the cytoplasm where it attaches with the 30S subunit of the ribosomes. The mRNA carries the triplet codons for the synthesis of the proteins. 2. The attachment of mRNA with 30s ribosomes and formation of polyribosome  In prokaryotic cells it has been observed that before the process of protein synthesis the ribosomes occur in dissociated and inactive state.
The mRNA binds with the 30S ribosomal subunit in the presence of IF2 protein factor. Soon the N-formlymethionine-tRNA (F-met tRNA') comes from the cytoplasmic "amino acid pool" and binds with the first triplet codon (5'-AUG-+3') of the mRNA to initiate the process of protein synthesis and to form the initiation complex. The formation of initiation complex is aided by the GTP (guanosine triphosphate) and 3 protein factors (F1, F2 and F3). After the formation of initiation complex the 30S ribosomal subunit unites with 50S ribosomal subunit to form the 70S ribosome. The union of ribosomal subunits occurs in the presence of Mg++ ions and F1 and F2 factors. The codes of mRNA are not read only by one ribosome but many ribosomes move and read the codes of the mRNA. When many ribosomes bind with the mRNA the formation of polysome or polyribosome occurs.
3. The transfer of amino acids to the site of protein synthesis   The amino acids are transferred from the intra-cellular amino acid pool to the active ribosomes by the tRNA. The transfer processes occur in various stages which are as follows: (a) Activation of amino acids-Each of the 20 amino acid occur in the cytoplasm in an inactive state. Each amino acid before its attachment with its specific tRNA is activated by a specific activating enzyme known as the aminoacyl synthetase and ATP.The free amino acid react with ATP, resulting in the production of aminoacyl adenylate and pyrophosphate :
1.DNA molecule 2.Transcription 3.mRNA 4.Nucleus 5.Cytoplasm 6.Unloaded tRNA 7.Loaded F-met tRNA 8.30S subunit of ribosome 9.50S subunit of ribosome 10.Initiation complex 11.Activated amino acid 12.Functional 70S ribosome 13.Amino acid pool     1.mRNA 2.peptidyl transferase 3.polypeptide chain
	The reaction product aminoacyl adenylate is bound to the enzyme in the form of a monocovalent complex. This aminoacyl adennylate enzyme complex then esterifies to specific tRNA molecule. The cell has at least 20 aminoacyl synthetase enzymes for the "0 amino acids. Each enzyme is specific and it attaches with the scientific amino acid without-any error. (b) Attachment of activated amino acid to tRNA - The amino adenylate remains bounded with enzyme until it collides with the specific tRNA molecule and its synthetase is recognized by dihydrouridine(DKU) loop of specific tRNA. Then, amino acid residue of aminoacyl adenylate is transferred to amino acid attachment site of tRNA where its carboxyl group forms bond or linkage with the 3-OH group of-the ribose of the terminal adenosine at CCA end of tRNA. As a result AMP and enzyme are released and a final product aminoacyl tRNA is formed by the following method:

The aminoacyl tRNA moves towards the site of protein synthesis. i.e., ribosomes with mRNA.
4. Initiation of protein synthesis
As we haft already mentioned that the initiation of protein synthesis in the bacterium Escherichia coli (E. coli) involves the formation 70S complex. In it the mRNA always; has, first triplet codon as AUG at its beginning (i.e., 5' end). The AUG codons are the codes for the amino acid methionine. The methionine remains formylated and it has a very important role in initiating the process of protein synthesis. In every type of protein the formyl methionine occupies the first place in the molecule and when the protein molecule is completely synthesized then the formyl methionine often detaches from the newly synthesized protein molecule by the activity of a hydrolytic enzyme.
Since in protein synthesis the peptide chain always grows in a sequence from the free terminal amino (-NH₂) group towards the carboxyl (-COOH) end, the function of formyl methionine-tRNA is to ensure that proteins are synthesized in that direction. In the formyl metionine-tRNA, the amino (-NH₂) group is blocked by the formyl group leaving only the -COOH group available to react with the-NH2 group of the second amino acid (AA₂). In this way the synthesis of protein chain follows in the correct sequence.
5. Elongation of polypeptide chain
With the formation of functional 70S ribosome (i.e., 70S-mRNA-F met tRNA), the elongation of polypeptide chain is brought, about by the regular addition of amino acids and relative movements of ribosome and mRNA in the presence of GTP molecules, so that a new triplet codon remains available for new aminoacyl tRNA at the decoding or 'A' site of ribosome in each step. Thus, F-met tRNA must move from decoding site ('A' site) to peptidyl site or 'P' site, before the second aminoacyl-tRNA (i.e., AA₂ tRNA) can bind to the next triplet condon occurring at decoding or 'A' site of ribosome. The aminoacyl t-tRNA (AA₂-tRNA) binds with the codon of 'A' site in the presence' of GTP and two proteins, called transfer factors (designed Tu and Ts) which remain associated with ribosome. During this binding process, a complex is formed between GTP, the transfer factors and the aminoacyl-tRNA (viz., AA-2 tRNA), which ultimately deposits aminoacyl-tRNA at the 'A' site of ribosome with the release of transfer factors GDP complex and inorganic phosphate.
In the next step, due to relative movement of ribosome and mRNA in the presence of EFG factor or translocase and GTP molecule, the next triplet codon becomes available for next aminoacyl tRNA (viz, AA,-tRNA) at the' A' site of ribosome. At this stage, f-met tRNA occurs at exit or 'E' site, while, AA2·tRNA occurs at peptidyl or 'P' site. Now, an enzyme known as transferase I kicks off tRNA from formyl methionine (f-met or AA1) and flips the formyl-methionine to the aminoacyl tRNA (AA.-tRNA) bound at the peptidyl or 'P' site. The 'G' factor is supposed to release the discharged or deacylated tRNA from the ribosome. The G factor is found to function in the presence of an acidic and contractile protein in the 50S ribosomal subunit (Kischa, et. al., 1971).
It follows the next stage of elongation process which involves the synthesis of a peptide bond by a· reaction (peptidyl transferase reaction) between the free amino group of the incoming amino acid (i.e., AA₂) and the carboxyl group of the first amino acid (AA₁) which is esterified to tRNA. The enzyme which catalyses this reaction is called peptidyl transferase (or peptide synthetase) and is an integral part of the 50S subunit. The energy for peptide bond synthesis is derived from cleavage of the ester link between an amino acid and its tRNA.
Thus, during the elongation of polypeptide chain each charged or loaded tRNA (aminoacyl tRNA) enters the decoding or 'A' site, moves to the condensing or 'P' site, transfers its amino acid to the carboxyl end of the polypeptide moves to the exit site, where the polypeptide chain is transferred to the adjacent tRNA, on the condensing site, and tRNA is then relased from the ribosome. This sequence of events involved in elongation must take place very rapidly since it has been calculated that in E. coli growing under optimal conditions, a polypeptide chain of about 40 amino acids can be produced in 20 seconds.
6. Termination and release of polypeptide chain
The synthesis of a polypeptide chain is concluded when a given ribosome in a polysome encounters a genetic signal encoded in the mRNA which specifies that the C-terminal amino acid of a polypeptide has been added to the chain. At least one of the termination signals is the presence of one or more terminator codons in the mRNA. A terminator codon is not recognized by the anti-codons of any of the normally occurring, aminoacyl-tRNAs and its presence in the decoding or aminoacyl site precludes the addition of any further amino acids to the chain. In E.coli, its phages and in eukaryotes, the RNA triplet UAA. UGA and UAG all function as terminator codons.
When a terminator codon moves into an amino acyl site, the following events are thought to occur. The terminator first, interacts with one of two specific protein factors called releasing factors (R₁ and R₂). The R₁ is specific for UAG and UAA and R2 for condons UAA and UGA. This complex of releasing factor terminator codon and ribosome effectively blocks further chain elongation. With the aminoacyl site so clogged, the completed polypeptide remains esterified to the final tRNA occupying the peptidyl site. This linkage is then broken by hydrolysis is a reaction mediated by still another protein factor, and both a free tRNA molecule and a complete polypeptide are released from the ribosome. The ribosome then dissociates into its large and small subunits, an event that may be mediated by F₃ protein factor. The dissociated subunits arc now free to form new initiation complexes and participate in another round of polypeptide synthesis.
7. Modification of released polypeptide
The released polypeptide chain contains the formylated methionine at its one end. An enzyme deformylase removes the formyl group of methionine. The exopeptidase enzyme may remove some amino acids from N-terminal end or the C-terminal end of polypeptide chain. At this stage the polypeptide (protein) possesses its primary and probably its secondary structures. The linear sequence of amino acids forms the primary structure; at least some portion of many proteins have a secondary structure in the form of an alpha-helix. The protein chain may then fold back upon itself, forming internal bonds (including strong disulfide bonds) which stabilize its tertiary structure into a precisely and often intricately folded pattern. Two or more tertiary structures may unite into a functional quarternary structure. For example haemoglobin consists of four polypeptide chains two identical (It chains and two identical p-chains. A protein does not become an activity enzyme until it has assumed its tertiary or quarternary pattern.

A. Primary structure	B.Secondary structure	C. Tertiary Structure	D. Active form of bovine pancreatic ribonuclease
E.Idealized structure of haemglobin containing 2 alpha and 2 beta chains	F.Quarternary structure	G.α-helix	H.Aminoacid sequence