The following points highlight the six main stages involved in protein synthesis of prokaryotic and eukaryotic systems. The stages are: 1. Amino Acid Activation and Formation of Amino Acyl-t-RNA 2. Binding of m-RNA to the Ribosome 3. Formation of Initiation Complex 4. Polypeptide Chain Elongation 5. Polysomes 6. Co Transcriptional Translation.
Stage # 1. Amino Acid Activation and Formation of Amino Acyl-t-RNA:
Amino acids require to be raised to a higher energy level to make them competent to be transferred to the t-RNAs. The activation of amino acids occurs by addition of AMP from ATP catalyzed by the enzyme amino acyI synthetase. The pyrophosphate group of ATP is released in the process. The same enzyme also catalyses the transfer of amino-acyI-AMP to its specific t-RNA. All protein amino acids are amino acids having the general structure.
The two-step reaction catalyzed by amino-acyI synthetase is:
The synthetase-bound amino acyl-AMP next reacts with its appropriate t-RNA producing amino acyl-t-RNA and AMP is set free. All t-RNA molecules possess a CCA sequence at the 3′-(OH) end. The amino acyl group of the amino acyl-AMP is transferred to the terminal adenylic acid of CCA sequence with the formation of a covalent linkage with either the 2′ or 3′ (OH) group of the ribose of adenylic acid as shown below (Fig. 9.41).
It should be specially mentioned that just as each amino acid is selected by its specific t-RNA, so also the formation of an amino acyl-t-RNA is catalysed by a specific amino acyl synthetase. A synthetase molecule can recognize its specific t-RNA with the help of the three-dimensional conformation of the protein molecule and that of the t-RNA.
It must also recognize the specific amino acid. Thus, in the intracellular pool, there are many different synthetase molecules, each of which is specific for both an amino acid and its cognate t-RNA. This one-to-one relationship is complicated by the presence of more than one t-RNA for most of the amino acids. To match the different codons of the same amino acid, there are different t-RNAs carrying complementary anticodons.
Once amino acyl-t-RNA has been formed, the amino acid plays no active role in selecting the site where it is to be inserted in the polypeptide chain, because it has no means to recognize the codon. It is carried passively by the t-RNA to its appropriate site. The t-RNA recognizes the m-RNA codon with its anticodon and brings the amino acid to its proper site.
Stage # 2. Binding of m-RNA to the Ribosome:
Protein synthesis does not take place on free m-RNA, but only on m-RNA bound to ribosomes. That is why ribosomes are sometimes referred to as ‘work-benches’ of protein synthesis. In both prokaryotes and eukaryotes, the m-RNA binds first to the small subunit of the ribosome i.e. the 30S subunit in prokaryotes and the 40S subunit in eukaryotes. The large subunit of the ribosomes is attached later to form the initiation complex.
In the prokaryotes, the m-RNA binds to 30S ribosomal subunit before the first amino acid is carried by the t-RNA. The first codon to initiate protein synthesis is AUG, but this initiator codon is preceded by a 20-30 nucleotide long sequence at the 5′-end of m-RNA. This means that the initiator codon (AUG) is situated 20-30 nucleotides downstream from the 5′-end. Within this preceding sequence, there is a short consensus sequence consisting of 5′–AGGAGGU-3′ situated 4 to 7 nucleotide ahead of the initiator codon AUG.
This consensus sequence is known as the Shine-Dalgarno sequence. This sequence helps in binding of m-RNA to the 30S subunit by forming base-pairs with its 16S r-RNA. At the same time, this sequence also acts as a signal for initiating protein synthesis at the next AUG sequence.
This is diagrammatically shown in Fig. 9.42:
The initiator codon codes for methionine, but all prokaryotic proteins have formyl-methionine (fmet) as the first amino acid at the amino terminus. In prokaryotes, t-RNAmet picks up methionine with the help of methionyl t-RNA synthetase and methionine is then formylated by another enzyme, transformylase, the donor of the formyl group being N10-formyl-tetrahydrofolate (formyl-THF). The formyl group is attached to the amino group of methionine.
The transfer reaction is:
Formylation of methionine can only occur when methionine is carried by t-RNAfmel and not when methionine is charged on t-RNAmet Thus, t-RNAfmet and t-RNAmcl are two distinct species although both are amino acylated by the same synthetase. The latter, i.e. t-RNA™’, carries methionine to the codon AUG situated inside the m-RNA but not to the initiator AUG codon.
In eukaryotic proteins, the first amino acid at the amino terminus is methionine and not formyl- methionine as in prokaryotes and the initiator codon is the same, i.e. AUG. However, in eukaryotes also, the initiator t-RNAmet and t-RNAmet for internal methionine are two distinct species. The former recognizes only the initiator AUG codon and no other AUG codons.
The initiator codon in eukaryotic m-RNA is situated 50 to 100 nucleotides downstream from the 5′ end. The 5′ end of eukaryotic m-RNA’s is always capped by methyl guanosine. This capped end binds to the 40S subunit of ribosome and the ribosomal subunit then moves along the m-RNA in the 5′ —> 3′ direction and scans the m-RNA triplets until it reaches an AUG sequence. At this point the initiation complex is formed, thereby also fixing the reading frame.
Stage # 3. Formation of Initiation Complex:
An initiation complex is an m-RNA-bound complete ribosome in which the initiator t-RNA carrying the first amino acid is attached and ready to receive the next incoming amino acid-charged t-RNA. In prokaryotes, formation of an initiation complex is preceded by the formation of a pre-initiation complex which is composed of the 30S subunit of the ribosome, the m-RNA molecule, a charged t-RNAfmet, three non-ribosomal proteins (initiation factors, IF) IF1, IF2 and IF3 and a molecule of GTP. The initiation factors, IF1 and IF3, help to dissociate the 70S ribosomes into 30S and 50S subunits, so that m-RNA can bind to the 30S subunit to form a pre-initiation complex.
The initiation factor IF2 mediates binding of GTP and the charged t-RNAfmet to the pre-initiation complex. Two other ribosomal proteins, SI and S12 are necessary for binding the m-RNA to the 16S r-RNA of the 30S subunit (Shine-Dalgarno sequence).
After the formation of the pre-initiation complex has been completed, the 50S subunit of the ribosome binds to it resulting in liberation of IF1. Next, IF2 is released by hydrolysis of GTP to GDP+Pi. With the attachment of the 50S subunit, the formation of initiation complex is completed.
The stepwise formation is shown in Fig. 9.43:
The binding of the 30S and 50S subunits creates two sites for binding of two t-RNA molecules. These are called the amino acyl site (A site) and the peptidyl site (P site). These sites overlap both the subunits of ribosome. The initiator, t-RNAfmet carrying formyl-methionine which was bound to the pre-initiation complex, is transferred to the P site of the initiation complex where its anticodon pairs with the initiator codon AUG of the m-RNA. The initiation complex is now ready for chain elongation.
So far as it is known, the sequence of events in the formation of the initiation complex in eukaryotes does not differ essentially from that of the prokaryotes, except that the number of initiation factors is more (at least nine) and binding of the m-RNA requires hydrolysis of ATP, a feature not known to occur in prokaryotes. In eukaryotes the initiator t-RNA carries methionine and not formyl-methionine as in prokaryotes. The initiator codon is the same i.e. AUG in both prokaryotes and eukaryotes.
Stage # 4. Polypeptide Chain Elongation:
In the initiation complex, the P site is occupied by the t-RNAfmet and the A site is vacant. It is now ready to be occupied by the incoming t-RNA carrying an appropriate amino acid. The anticodon of this t-RNA must match with the triplet of m-RNA positioned at the A site. The binding of the t-RNA to the A site requires a non-ribosomal cytoplasmic protein factor, called the elongation factor Tu (EF-Tu) which is activated by GTP.
The three components, viz. t-RNA, EF-Tu and GTP, form a ternary complex which binds to the A site of the ribosome. In this binding process, the TᴪC arm of t-RNA (see structure of t-RNA) is thought to interact with the 5S r-RNA of the 50S subunit of the ribosome. Once the charged t-RNA (incoming) binds firmly to a site of the ribosome, GTP is hydrolysed by a ribosomal protein enzyme to release GDP-EF-Tu and inorganic phosphate.
The A site is now occupied by the incoming t-RNA carrying the second amino acid. From the GDP-EF-Tu binary complex, GTP-EF-Tu is regenerated by another protein factor, called EF-Ts and inorganic phosphate. GTP-EF-Tu can then combine with the next incoming aminoacyl t-RNA to form the ternary complex.
The sequence of events are diagrammatically represented in Fig. 9.44:
The second step in the chain elongation process involves the formation of a peptide bond between the α-amino group the amino acid occupying the A site and the α-carboxyl group of the amino acid occupying the P site.
However, the peptide bond formation takes place by a complex reaction, because the carboxyl group of the amino acid in amino acyl-t-RNA at the P site is not free, but linked to the adenylic acid residue of CCA sequence of t-RNA (see Fig. 9.41).
The reaction is called peptidyl transferase reaction. It takes place in the 50S subunit of the ribosome. As a result of the reaction, the P site is now occupied by an uncharged t-RNA (without amino acid) and the A site is occupied by a t-RNA with a dipeptide as shown in Fig. 9.45.
In the next step, the t-RNA with the bound dipeptide moves from the A site to the P site displacing the empty t-RNAfmet. The movement is known as translocation and it is catalysed by another elongation factor EF-G which binds to the ribosome. In the process, hydrolysis of GTP catalysed by a ribosomal protein is known to occur.
Concurrent with translocation from the A site to P site, the m-RNA also moves through one coding unit in the 3′ —> 5′ direction. Thereby, the next codon is positioned at the A site. These processes of peptidyl transferase reaction and translocation take place with addition of each amino acid carried by the respective t-RNAs. As a result, the polypeptide chain elongates at the amino-terminal end by sequential addition of amino acids one by one determined by the matching of t-RNA anticodon and m-RNA codon.
The final step of protein synthesis is reached when, at the A site, one of the three termination codons (UAA, UAG or UGA) of m-RNA appears. As these codons do not code for any amino acid (that is why they are called nonsense codons), the A site remains vacant. The polypeptide chain is released from the ribosome through the action of a release factor (RF).
In prokaryotes, there are three release factors, RF1, RF2, and RF3. They bind to the ribosome and catalyse hydrolysis of the ester linkage between the t-RNA occupying the P site and the carboxyl group of the last amino acid. Thereby the polypeptide chain becomes free to be released from the ribosome. The 70S ribosome then falls off from the m-RNA.
Stage # 5. Polysomes:
In considering protein synthesis above, the mutual relation between an m-RNA and a single 70S ribosome has been dealt with. However, in practice, a single m-RNA molecule is used for multiple translation in both prokaryotes and eukaryotes. Such a practice is economical for the cell, because several copies of the same protein can be produced in a relatively short time without going through the process of transcription.
When the chain elongation phase of polypeptide synthesis has advanced to a stage, so that the chain is 25-30 amino acid long, the initiator AUG codon of the m-RNA becomes free to form another initiation complex in the same way as it formed the first one. So, a second 70S ribosome initiates synthesis of a second copy of the same polypeptide.
In this way initiation may be repeated several times on different ribosomes, thereby forming a string of ribosomes bound by a common m-RNA molecule. Such a structure is called a polyribosome or polysome. The size of a polysome varies according to the length of the m-RNA molecule.
There may be 3 to 4 or as many as 100 ribosomes in a polysome. It is obvious that polypeptide synthesis terminates in the first ribosome and then in the second and so an. At any given time, the length of the polypeptide chains varies depending on the progress of chain elongation in the ribosomes of a polysome complex.
A diagrammatic representation of polypeptide synthesis in a polysome is shown in Fig. 9.46:
Stage # 6. Co Transcriptional Translation:
In eukaryotes, the primary transcript, known as hn-RNA has to be processed by removing the introns, capping the 5′-end and adding a poly A-tail at the 3′ end. The processed product, the m-RNA, is then trans-located from the nucleus into the cytoplasm through pores in the nuclear membrane. In contrast, the primary transcript in prokaryotes is used directly as the m-RNA.
Also, the nuclear material is not separated by a membrane from the cytoplasm. A characteristic feature of the prokaryotic protein synthesis is, therefore, that translation of the m-RNA can begin while the m-RNA is still being transcribed from the template strand of DNA. This is known as simultaneous transcription and translation, or co-transcriptional translation.
As the RNA polymerase moves along the DNA template strand, 5′ end of the m-RNA comes out and binds to a 30S subunit by base-pairing with the Shine-Dalgarno sequence. An initiation complex is formed in the usual way and polypeptide synthesis begins. With progress of transcription, the m-RNA grows in length and it can bind more ribosomes forming a polysome (Fig. 9.47).
The phenomenon of co-transcriptional translation in prokaryotes assumes special significance in view of the fact that prokaryotic messengers are very short-lived having an average half-life only 1.3 to 1.8 minutes. Simultaneous transcription and translation can, therefore, make best use of a messenger molecule by shortening the two processes through coupling them.
In E. coli the rate of transcription at 37°C is 55 nucleotides/sec and that of translation is 17 amino acids per second. Therefore, transcription which is faster can run concurrently with the slower process of translation. Thereby the total time for protein synthesis can be considerably reduced.