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The following points highlight the three main steps involved in mechanism of protein synthesis. The steps are: 1. Initiation of Protein Synthesis 2. Elongation of Polypeptide 3. Termination of Protein Synthesis.
Mechanism of Protein Synthesis: Step # 1. Initiation of Protein Synthesis:
(i) Amino Acid Activation:
The amino acid (AA) is activated by the enzyme “aminoacylsynthetase”, and energy is provided by breakage of ATR The -OH group of carboxyl end of the amino acid and -OH group of phosphate of AMP react to form amino-acyladenylate (AA-AMP), releasing a molecule of pyrophosphate (Fig. 4.5):
(ii) Transfer of Amino Acid to tRNA:
The amino-acyladenylate (AA-AMP) is bound tightly with the enzyme aminoacylsynthetase. The same enzyme catalyses the reaction of transfer of the amino acid to tRNA ;aminoacyl-tRNA (AA~ tRNA) is formed and AMP is released :
The amino acid is attached by its -OH group of carboxyl to the -OH group of 3′ position of ribose sugar of tRNA (Fig. 4.5). Thus the amino acid associated at the 5′-P end of AMP becomes associated with the 3′ position of sugar in the adenine nucleotide of tRNA. The enzyme aminoacylsynthetase possesses two different active sites, the site for recognition of side group of an amino acid, and the site for recognizing the tRNA specific for the amino acid.
(iii) Binding of mRNA to Small Subunit of Ribosome:
In prokaryotes, the initiation factor 3 (IF3) binds to 30S ribosomal subunit to form an “IF3-30S” complex. This complex binds to mRNA to form the IF3-30S mRNA complex. The initiation factor 2 (IF2) gets attached to N-f. met-tRNAfmet and helps to bind to the above complex.
A molecule of GTP is associated with the initiation complex, it provides energy for association of 50S particle with the above complex to make a 70S ribosome. The IF1 helps in this reaction indirectly. GTP is hydrolysed to GDP and initiation factors are released (Fig. 4.6).
In eukaryotes, the first amino acid is methionine and it is not formylated. Initiation factors are greater in number than in prokaryotes. GTP associates with initiation factor elF2 and makes a complex with the amino acylt RNA. This complex becomes associated with the small ribosomal subunit (40S). Then the 5′ -end of mRNA binds to the 40S initiation complex with the help of several initiation factors.
The initiation complex moves in 3′ direction to locate the AUG codon. At the same time, 60S subunit gets associated with the 40S subunit to form an 80S ribosome. GTP is hydrolysed to GDP for providing energy to this reaction.
Mechanism of Protein Synthesis: Step # 2. Elongation of Polypeptide:
The N-formyl-methionyl-tRNA (in prokaryotes) or methionyl-tRNA (in eukaryotes) occupies the “P” site on the ribosome.
Once an initiation complex is formed, newly initiated polypetide chain is elongated by regular addition of amino acids, one-by-one, in an order specified by the codon in mRNA.
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The process of elongation involves three steps:
(i) Binding of AA~tRNA to the ribosome,
(ii) Peptide bond formation, and
(iii) Translocation.
(i) Binding of AA~tRNA to Ribosome:
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With the binding of AA~tRNA to the “A” site of the ribosome starts the first elongation cycle. The selection of AA~tRNA is made according to the “anticodon-codon” pairing. In prokaryotes, a protein EF-Tu (elongation factor Tu) and GTP are required for binding the AA-tRNA to ribosomal “A” site. The EF-Tu promotes the binding of all AA~ tRNAs to ribosome except the initiator (N-f. met-tRNAfmet).
But these factors cannot recognize specific anticodon. There exists some mechanism that ensures the entry of correct AA -tRNA at the “A” site. GTP is hydrolysed to GDP and the complex EF-Tu-GDP is released. This complex is converted into EF-Tu-GTP with the help of another elongation factor “EF- Ts”. In eukaryotes, a single factor EFI performs the function of both EF-Tu and EF-Ts.
(ii) Peptide Bond Formation:
Peptide bond formation occurs between the free amino group (-NH2) of amino acid attached to tRNA at “A” site and carboxyl group 
by which the initiating amino acid (or growing polypeptide chain) is attached to the tRNA at “P” site. Thus the newly formed peptide is transferred to the “A” site and the tRNA at “P” site becomes free. The enzyme involved is peptidyltransferase.
(iii) Translocation:
Protein synthesis starts at 5′-end of mRNA and advances towards 3′- end The movement of mRNA occurs in 3’—>5′ direction. After peptide bond formation, the tRNA at “A ” site carries the polypeptide .chain and called the “peptidyltRNA”.
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The mRNA advances by one triplet unit (codon) to bring the next codon into accurate position. With the movement of mRNA, the peptidyl-tRNA also moves from “A” to “P” site displacing the empty tRNA from the ribosome (Fig. 4.7). This process is called “translocation” and its requires GTP and elongation factor EF-G in prokaryotes (EF2 in eukaryotes).
Now a new AA ~ tRNA binds to the “A” site and a new elongation cycle begins.
Mechanism of Protein Synthesis: Step # 3. Termination of Protein Synthesis:
There are 3 codons, namely UAA, UAG and UGA which do not specify some amino acid. They are utilized by the organism as “termination codons” or “stop codons” for growing polypeptide chain. The mRNA moves in 3’—>5′ direction and amino acid is added one-by-one. When the stop codon comes at the ribosome, no AA-tRNA comes on “A” site, and therefore, incorporation of amino acid in the polypeptide chain stops.
In prokaryotes, termination codons are recognized by 3 proteins called “release factors” (RF1, RF2, RF3,). The RF1 binds to UAA and UAG, while the RF2 binds to UAA and UGA. The RF3 stimulates the activity of both the factors RF1 and RF2. In eukaryotes, a single release factor eRFl recognizes all the termination codons.
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The linkage between the tRNA and polypeptide is hydrolysed and polypeptide chain separates; mRNA, tRNA are released from the ribosomes that are dissociated into subunits (Fig. 4.7). The termination process requires GTP which is hydrolysed into GDP.
Polyribosomes or Polysomes:
After beginning of protein synthesis, the 5′-end of mRNA becomes free as it moves. Then another ribosomal subunit binds to mRNA at the 5′-end and a new initiation cycle starts. In this way, several ribosomes get associated with the same mRNA, each synthesizing a polypeptide chain.
This complex structure is called “polyribosomes”. Number of ribosomes in the cluster (polyribosomes) depends on the length of mRNA. There is one ribosome for 80 nucleotides of mRNA for the maximum utilization of mRNA.
For haemoglobins, 4-6 ribosomes make a polyribosomal complex. The mRNA producing a polypeptide chain of about 300 amino acids will be occupied by about 12 ribosomes. For very large proteins, there may be 50 or more ribosomes on the same mRNA.
By this mechanism, the efficiency of utilization of mRNA is increased and many polypeptide chains are produced. However, the polypeptide chains on different ribosomes complete at different times.
Changes in the Newly Produced Polypeptide:
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The newly released polypeptide carries N- formyl-methionine at one end and the carboxyl group at the other end. The formate group is removed by the enzyme deformylase, while the methionine (initiator amino acid) is removed from the amino terminal end by the enzyme “methionine specific amino peptidase”.
Rates of Transcription and Translation:
The rate of transcription in prokaryotes (bacteria) and eukaryotes is similar; it is about 40 nucleotides per second at 37°C. Thus the time taken for synthesis of myRNA can be calculated if the length of the gene is known. A 5,000 bp long DNA would take about 2 minutes to be transcribed.
The rate of protein synthesis (translation) in bacteria is nearly the same as the rate of transcription, i.e., roughly 45 nucleotides (corresponding to 15 amino acids) are translated per second. But in eukaryotes,) the translation speed is slower; it occurs at a rate of 20 nucleotides per second.
Life of a bacterial mRNA is very short; its degradation starts from 5′-end even before the 3′-end has been synthesized. Half-life of bacterial mRNA is about 2 minutes. Eukaryotic mRNAs are protected by 5′-capping and 3′-adenylation and therefore, life of an eukaryotic mRNA is quite long; it ranges from 4 to 24 hours in animal cells.
