Translation is the mechanism by which the triplet base sequence of a mRNA guides the linking of a specific sequence of amino acids to form a polypeptide (protein) on ribosomes.
Protein synthesis requires amino acids, DNA, RNAs, ribosomes and enzymes. The mechanism of protein synthesis involves four steps. The four steps are: (1) Activation of Amino Acids (2) Charging of tRNA (3) Activation of Ribosomes and (4)Assembly of Amino Acids (Polypeptide Formation).
Machinery for Protein Synthesis:
Protein synthesis requires amino acids, DNA, RNAs, ribosomes and enzymes.
I. Amino Acids:
Proteins are the polymers of amino acids. Therefore, amino acids form the raw material for protein synthesis. The proteins of living organisms need about 20 amino acids as building blocks or monomers. These are available in the cytoplasmic matrix as an amino acid pool.
II. DNA as Specificity Control:
A cell, in order to maintain its own special characteristics, must manufacture proteins exactly similar to those present already in it. Thus, protein synthesis requires specificity control to provide instructions about the exact sequence in which the given numbers and kinds of amino acids should be linked to get the desired polypeptides.
The specificity control is exercised by DNA through mRNA sequences of 3 consecutive nitrogenous bases in the DNA double helix form the biochemical or genetic code. Each base triplet codes for a specific amino acid. Since the DNA is more or less stable, the proteins formed in a cell are exactly like the preexisting proteins.
III. RNAs:
RNA molecule is a long, un-branched, single-stranded polymer of ribonucleotides (Fig. 7.12). Each nucleotide unit is composed of three smaller molecules: a phosphate group, a 5- carbon ribose sugar, and a nitrogen-containing base. The bases in RNA are adenine, guanine, uracial and cytosine. The various components are linked up as in DNA.
There are three types of RNA in every cell: messenger RNA or mRNA, ribosomal RNA or rRNA and transfer RNA or tRNA. The three types of RNAs are transcribed from different regions of DNA template, RNA chain is complementary to the DNA strand which produces it. All the three kinds of RNAs play a role in protein synthesis.
(a) mRNA:
The DNA, that controls protein synthesis, is located in the chromosomes within the nucleus, whereas the ribosomes, on which the protein synthesis actually occurs, are placed in the cytoplasm. Therefore, some sort of agency must exist to carry instructions from the DNA to the ribosomes. This agency does exist in the form of mRNA.
The mRNA carries the message (information) from DNA about the sequence of particular amino acids to be joined to form a polypeptide, hence its name. It is also called informational RNA or template RNA. The mRNA forms about 5% of the total RNA of a cell. Its molecule is linear and the longest of all the three RNA types. Its length is related to the size of the polypeptide to be synthesized with its information.
There is a specific mRNA for each polypeptide. Because of the variation of size in mRNA population in a cell, the mRNA is often called heterogeneous nuclear RNA, or hn RNA:
In eukaryotes, mRNA carries information for one polypeptide only It is monocistronic (monogenic) because it is transcribed from a single cistron (gene) and has a single initiator codon and a single terminator codon.
Bacterial mRNA often carries information for more than one polypeptide chains. Such a mRNA is said to be polycistronic (polygenic) because it is transcribed from many contiguous (adjacent) genes. A polycistronic mRNA has an initiator codon and a terminator codon for each polypeptide to be formed by it.
(b) tRNA:
The tRNA has many varieties. Each variety carries a specific amino acid from the amino acid pool to the mRNA on the ribosomes to form a polypeptide, hence its name. The tRNAs form about 15% of the total RNA of a cell. Its’ molecule is the smallest of all the RNA types.
A tRNA molecule has the form of a clover leaf. It has four regions:
(i) Carrier End:
This is the 3′ end of the molecule. Here a specific amino acid joins it. It in all cases has a base triplet CCA with – OH at the tip. The – COOH of amino acid joins the – OH.
(ii) Recognition End:
It is the opposite end of the molecule. It has 3 unpaired ribonucleotides. The bases of these ribonucleotides have complementary bases on the mRNA chain. A base triplet on mRNA chain is called a codon, and its complementary base triplet on tRNA molecule is termed an anticodon. Anticodon reads its appropriate codon and temporarily joins it by hydrogen bonds during protein synthesis.
(iii) Enzyme Site:
It is on one side of the molecule. It is meant for a specific charging enzyme which catalyzes the union of a specific amino acid to tRNA molecule.
(iv) Ribosome Site:
It is on the other side of the molecule. It is meant for attachment to a ribosome.
(c) rRNA:
The rRNA molecule is greatly coiled. In combination with proteins, it forms the small and large subunits of the ribosomes, hence its name. It forms about 80% of the total RNA of a cell. The rRNA also seems to play some general role in protein synthesis.
(IV) Ribosomes:
Ribosomes serve as the site for protein synthesis. The small and large subunits of ribosomes occur separately when not involved in protein synthesis. The two sub units form association (join) when protein synthesis starts, and undergo dissociation (separate) when protein synthesis stops. Many ribosomes line up on the mRNA chain during protein synthesis. Such a group of active ribosomes is called a polyribosome, or simply a polysome.
In a polysome, the adjacent ribosomes are about 340 Å apart. The number of ribosomes in a polysome is related to the length of the mRNA molecule, which reflects the length of the polypeptide to be synthesized. It has been established that polypeptides are synthesized at the polysomes and not at the single free ribosomes as held earlier. This is true for both prokaryotes and eukaryotes as well as for the cell organelles such as mitochondria and plastids.
A ribosome has two binding sites for tRNA molecules. One is called A (acceptor or aminoacyl) site and the other is termed P (peptidyl) site. These sites span across the large and small subunits of the ribosome (Fig. 7.13). The A site receives the tRNA-amino acid complex. From P site, the tRNA leaves after leaving its amino acid to the forming polypeptide. However, the first tRNA-amino acid complex directly enters the P site of the ribosome.
The function of the ribosome is to hold in position the mRNA, tRNA and the associated enzymes controlling the process until a peptide bond forms between the adjacent amino acids.
Mechanism of Protein Synthesis:
Protein biosynthesis involves following major steps:
(i) Activation of Amino Acids:
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 synthetize 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 (Fig. 7.14). The pyrophosphate is hydrolysed to 2Pi, driving the reaction to the right.
(ii) Charging of tRNA:
The amino acid-AMP- enzyme complex joins to the amino acid binding site of its specific tRNA, where its -COOH group bonds to – OH group of the terminal base triplet CCA. The reaction is catalyzed by the same aminoacyl-tRNA synthetase enzyme.
The resulting tRNA-amino acid complex is called a charged tRNA (Fig. 7.14). AMP and enzyme are freed. The freed 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 on ribosomes.
The tRNA-amino acid complex moves to the site of protein synthesis, the ribosome.
(iii) Activation of Ribosomes:
The small and the large subunits of ribosomes must be joined together for protein synthesis. This is brought about by mRNA chain. The latter joins the small ribosomal subunit by first codon through base pairing with appropriate sequence on rRNA. The combination of the two is called initiation complex (Fig. 7.15). The large subunit later joins the small subunit, forming active ribosome. Activation of ribosome by mRNA requires proper concentration of Mg2+ (0.001 Molar conc.)
(iv) Assembly of Amino Acids (Polypeptide Formation):
The events in protein synthesis are better known in bacteria than in eukaryotes. Although these are thought to be similar in the two groups, some differences do occur. The following description refers mainly to protein synthesis in bacteria on the 70S ribosomes. Polypeptide formation involves 3 events: initiation, elongation and termination of amino acid chain.
(a) Initiation of Polypeptide Chain:
The mRNA chain has at its 5′ end an “initiator” or “start” codon (AUG) that signals the start of polypeptide formation. This codon lies close to the P site of the ribosome. The amino acid formyl-methionine (methionine in eukaryotes) initiates the process. It is carried by tRNA having UAC anticodon which bonds to AUG initiator codon of mRNA by hydrogen bonds.
Initiation factors (IF 1, IF 2 and IF 3) and GTP promote the initiation process. The large ribosomal subunit now joins the small subunit to complete the ribosome. At this stage, GTP is hydrolyzed to GDP. The ribosome has formylmethionine-bearing tRNA (tRNA fMet) at the P site (Fig. 7.15). Later, the formylmethionine is changed to normal methionine by the enzyme deformylase. If not required, methionine is later separated from the polypeptide chain by a proteolytic enzyme amino peptidase.
Initiation factors are used again to start new chains. As already established, translation of the codons of mRNA takes place in the 5′ – 3′ direction, thus P site and A site on the ribosomes recognize the polarity of the mRNA chain.
(b) Elongation Phase:
At this point fmet- tRNAfmet molecule in the 70S initiation complex occupies the P site on the ribosome. The other site for a tRNA molecule, i.e. the A site, is empty. The fmet-tRNAfmet is positioned in such a way that its anticodon pairs with the initiating AUG (or GUG) codon on mRNA. The reading frame is specified by this interaction and by pairing of the adjoining purine-rich sequence to a pyrimidine-rich sequence in 16S rRNA.
The elongation cycle in the protein synthesis begins with the insertion of an aminoacyl tRNA into the empty A site on the ribosome. The species of tRNA to be inserted depends upon the mRNA codon that is present in the A site. The complementary aminoacyl tRNA is transferred to the A site by a non-ribosomal specific cytoplasmic protein, called the elongation factor T (EF-T) that binds to the aminoacyl tRNA.
The factor EF-T contains two subunits, EF-Ts and EF-Tu. EF-Tu like IF2 contains a bound guanyl nucleotide and cycles between a GTP and a GDP. If the codon matches the anticodon, GTP is hydrolysed, positioning the aminoacyl tRNA in the A site and GDP bound with EF-Tu dissociates from the ribosome. A second elongation factor EF-Ts joins the EF-Tu complex and GDP is displaced from the complex forming a EF-Tu-Ts complex.
Finally, GTP binds to the EF-Tu- EF-Ts complex, releasing EF-Ts. EF-Tu containing bound GTP is ready to pick up another aminoacyl tRNA and deliver to the A site of the ribosome. This GTP-GDP cycle keeps repeating. It should be noted that EF-Tu does not recognise the fmet-tRNA initiator, hence the initiator tRNA is not delivered to the A site.
On the contrary before fmet-tRNAet, like all other aminoacyl tRNAs, can bind to EF-Tu. This explains why internal AUG codons are not read by initiator tRNA. It has been observed that rapid binding of EF-Tu to an activated aminoacyl-tRNA prevents hydrolysis, but after the formation of H’-Tu-GTP-tRNA complex, a time lag of several milliseconds allows the codon-anticodon mismatches to diffuse away (before OTP hydrolysis).
Peptide Bond Formation and Translocation:
Once the initiator fmet-tRNA occupies the P site and the next aminoacyl-tRNA occupies the A site, a peptide bond between the adjacent amino acids is formed by an enzyme, peptidyl transferase belonging to the 50S subunit. The active site of the peptidyl transferase is the 23 S rRNA. The uncharged tRNAfmet occupies the P site and the dipeptide formed is attached to the second tRNA occupying the A site following the formation of a peptide bond. The product of the first peptide bond formation is called dipeptidyl-tRNA bound to the A site.
The next step of the elongation cycle is translocation, which requires a third elongation factor EF-G (also called translocase) causing hydrolysis of GTP.
Three important movements occur:
(1) The fmet-tRNA which is now uncharged leaves the P site,
(2) The second tRNA with bound dipeptide is moved to the P site, and
(3) mRNA moves a distance of three nucleotides.
After translocation, the A site is opened up to accept the incoming aminoacyl-tRNA to match the next codon, now positioned at the A site for the next round of elongation (Fig. 7.16). The factor EF-Tu delivers the next aminoacyl-tRNA for the empty A site.
The accuracy of protein synthesis depends on having the correct aminoacyl-tRNA in the A site when the peptide bond is formed, hence the incoming aminoacyl-tRNA is meticulously scrutinized so that its anticodon is complementary and matches the codon at the A site. A mismatch aminoacyl- tRNA may bind with two or three nucleotides of a codon only temporarily, but will leave the A site before a peptide bond is formed. It takes a few milliseconds for the ribosome to decide if the incoming aminoacyl-tRNA is the correct one or not and the time lag is determined by GTPase site of EF-Tu. A peptide bond cannot be formed until EF-Tu is released from the aminoacyl-tRNA and the process requires hydrolysis of GTP to GDP and Pi.
(c) Termination Phase:
Two conditions are necessary for termination of protein synthesis. One is the presence of a stop codon that signals the chain elongation to terminate, and the other is the presence of release factors (RF) which recognise the chain terminating signal. There are three terminating codons, UAA, UGA and UAG for which tRNAs do not exist. Termination of polypeptide chain is signaled by one of these codons in the mRNA. Behind all this complexity is the fact that after the polypeptide chain has reached its full length, its carboxyl end is still bound to its tRNA adapter.
Termination must, therefore, involve the splitting of the terminal tRNA. Release of the peptidyl tRNA from the ribosome is promoted by three specific release factors, RF1, RF2 and RF3. RF1 recognises triplets UAA and UAG, while RF2 recognises UAA and UGA. The third factor RF3 does not possess any release activity of its own, but it binds to OTP and stimulates the binding of RF1 and RF2 with the ribosome.
In E. coli, 16S rRNA is essential in reading the stop codon. The release factors bind to stop codon to cause a shift of the polypeptidyl-tRNA from A to P site (Fig. 7.17). Whether OTP hydrolysis is required for chain termination is not yet firmly established, although the RF3, which appears to enhance RF1 and RF2 binding with ribosome, does not bind to OTP.
The ester bond between the polypeptide chain and the last tRNA is then hydrolysed. Binding of RF to the terminating codon causes water to act as the acceptor of the growing peptide and not another amino acid on a tRNA Release of the polypeptide chain is followed by dissociation of mRNA and tRNA. Subsequently dissociation of 30S and SOS ribosome subunits takes place with concomitant binding of IF3 to 30S subunit to prevent reassembly in the absence of mRNA and fmet-tRNA.
Modification of Released Polypeptide:
The just released polypeptide has primary structure, i.e., it is a straight, linear molecule. It is often called as nascent polypeptide. It may lose some amino acids from the end with the help of an exopeptidase enzyme, and then coil and fold on itself to acquire secondary and tertiary structure. It may combine with other polypeptides to have quaternary structure. 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.
Polysome Formation and Translational Amplification:
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 copy 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 stop codon.
A row of ribosomes joined to the mRNA molecule, is called a polyribosome, or simply a polysome. Synthesis of many molecules of the same polypeptide simultaneously from one mRNA molecule by a polysome is called translational amplification.
Inhibitors of Protein Synthesis in Prokaryotes:
Antibiotics are the bio-chemicals synthesized by bacteria and some fungi. Many antibiotics are known to block the bacterial translocation. This forms the basis of checking bacterial infection without harming the human host.
Non-ribosomal Protein synthesis:
Recently, the biosynthesis of many polypeptide antibiotics are known to be mediated by soluble enzymes of bacterial cells rather than by ribosomes. e.g., gramicidin S, gramicidin A, actinomycin D etc.