The protein biosynthesis process involves following steps among which one step i.e. activation of aminoacid is discussed in previous article.
In 1961, Howard Dintzis suggested that protein biosynthesis starts at the amino terminal end and followed by incorporation of amino acids to the carboxylic acid and of the growing poly peptide chain. The initiation process required initiation codon AUG which specifies Methionine. All organisms have two tRNA for Methionine but codon i.e. AUG is same. One tRNA is utilized when AUG acts as initiation codon and second tRNA us used when AUG is present in internal position in Polypeptide chain. In bacteria methionine present at initial position get formylated. The tRNA used for carrying formylated methionine is designated as tRNA f met. The formyl group attached to the methionine by two successive steps
a) Methionine is attached to the tRNA f met with the help of enzyme Met- tRNA synthetase. The reaction is as follows:
Methionine + tRNA fmet + ATP = Met-tRNA f met + AMP + PPi
b) A highly selective enzyme Transformylase transfers a formyl group from N10formyltetrahydrofolate to methionine residue present at initial position.
N10-formyltetrahydrofolate + met-tRNA f met = tetrahydrofolate + fMet- tRNA f met
N formyl group attached to the methionine allow fMet-tRNA bound to the specific ribosomal initiation site. This initiation process requires set of three cytosolic proteins known as initiation factors (IF-1, 2 and 3), the 30S and 50S ribosomal subunit, mRNA. The initiation process starts with the binding of two initiation factor IF-1 and IF-3 to the 30 S ribosomal subunit. Factor IF3 prevents the premature binding of 30 S and 50 S subunit. After that mRNA binds to 30 S subunit. The initiation codon AUG is recruited on ribosome to its correct position by shine-Dalgarno sequence which is an initiation signal of 4 to 9 purine residue present at 8 to 18 base pair away from the initiation codon. This sequences consensus sequence which base pair with 16 S rRNA of 30 S ribosomal subunit. Bacterial ribosome consists of 3 sites; amino acyl (A) site, the peptidyl (P) site and the exit (E) site. The initiation codon AUG present on P site to which only fMet-tRNA fMet can bind firstly. All incoming tRNA loaded with amino acid bind to the A site firstly then to the P site and E site. The E site as name implies is the site from which uncharged tRNA are released during elongation. Factor IF-2 binds to the A site and inhibit the binding of other incoming amino acyl - tRNA during initiation. IF2 along with f met tRNA join the complex which already contains 30 S ribosomal subunit, mRNA, IF1 and IF3. This large complex combined with 50 S subunit of ribosome. Meanwhile GTP is hydrolyzed to GDP and Pi and IF1, 2, 3 are released form the complex. This functional 70 S ribosome is known as initiation complex which consists of 50 S ribosomal subunit, 30 S subunit, mRNA and f met tRNA f met.
In bacteria elongation process requires following components. 1) Initiation complex
2) Amino acyl tRNA 3) Elongation factors (EF-Tu, EF-Ts and EF - G) 4) GTP.
This process completes in 3 steps which are discussed below.
Step 1: Correct incoming amino acyl tRNA binds to the complex of GDP bound EF - Tu (Elongation factor temperature unstable). Amino acyl tRNA GTP EF - Tu complex binds to the A site. The EF-Tu GDP complex released from the 70 S ribosome after the hydrolysis of GTP. EF-Tu then binds to the EF-Ts (Elongation factor temperature stable). Then EF - Tu binds to GTP. This EF - Tu - Ts cycle recycles EF - Tu GTP complex and makes available EF - Tu GTP which is ready to carry another amino acyl tRNA.
Step 2: During this phase of elongation a peptide bond is formed between two amino acids present on A & P site of ribosome. An enzyme known as peptidyl transferase present on large ribosomal unit catalyzes the transfer of N formyl methonyl group from its RNA to the amino group of second amino acid present on A site producing di peptidyl tRNA in A site and leaving uncharged tRNA on P site.
Step 3: The final step of elongation is translocation. The ribosome moved one codon away towards 3' end of mRNA. This movement causes di peptidyl tRNA to shift at P site, uncharged tRNA to E site from where tRNA gets released. Now the A site is empty for another amino acyl tRNA. This translocation is facilitated by EF - G translocase and requires GTP to meet the requirement of energy. Now dipeptidyl tRNA with ribosome and mRNA is ready for another elongation cycle.
Protein biosynthesis process comes to termination when A site of ribosome encounter with one of the three termination codon on mRNA. UAA, UGA and UAG are considered as termination codon.There are three termination factors known as release factors RF-1 RF-2 and RF-3 which causes termination of the process. RF-1 recognizes UAG and UAA, RF-2 recognizes UGA and UAA. The specific function of RF - 3 is not established yet but known to release the ribosome into its subunits. Binding of RF into the termination codon results into the hydrolysis of peptidyl tRNA on P site, release of polypeptide chain and release of uncharged tRNA from P site.
In this way the polypeptide chain is synthesized but in order to attain the biologically active form the polypeptide chain must go through the process of protein folding and post translational modification.
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