The Biology Topics of ecology involve studying the relationships between living organisms and their environment.
Translation Protein Synthesis – Structure and Role of tRNA
Proteins play an important role in the construction of the body structure as well as in the gene expression of living beings. In the organism major part of the body components is protein by nature and the enzymes carrying out almost all biological activities are also proteins. During the life period, an organism may produce different protein elements as required from time to time. Every protein actually represents a chain of amino acids, the polypeptide. The polypeptide chain is the primary configuration of a protein in which the amino acids may be linked in the linear array by an organized system within the cell. This primary polypeptide chain may achieve tertiary or quarternary configuration in order to produce a functional protein molecule.
The process by which a cell may produce a polypeptide chain is called translation. The part of the cellular DNA meaning a gene is actually responsible for the synthesis of a specific polypeptide chain. A genic segment of DNA may be called a store of information and that information is transferred to mRNA in coded form therefore, the RNA produced from a gene segment is called messenger RNA. The coded message in mRNA may be expressed as a chain of amino acids in this process and therefore, the process is termed as translation. As if, in the process, one language is converted into another. The proteins produced in this way become the ultimate fate in the gene expression. The polypeptide chain may be converted into protein through post-translational processing.
In all living organisms the mechanism of translation is identical and most of the knowledge about this we have obtained from studies on E. coli. The 1 mm long DNA of E. coli contains about four thousand genes and most of the genes are transcribed into mRNA and each mRNA produces polypeptide chain through sequential steps.
Requirements for Translation
mRNA is the primary requirement for the polypeptide chain synthesis and without the mRNA molecule, the production of the polypeptide chain is practically impossible. Along the entire length of the mRNA, there are many codons and according to codons in sequence, amino acids may be incorporated into a polypeptide chain. In the mRNA from 5′ end to 3′ end all the codons together are messages from the gene and this message by way of translation is converted into a polypeptide chain.
2. Amino Acids:
Altogether 20 amino acids are involved in the formation of protein. They are present in the cellular pool in the cytoplasm and may be collected from this pool as per the requirement so that they may be utilized for their polymerization into a polypeptide chain.
- Amino acyl tRNA Synthetase: This enzyme carries out two functions. On one hand, it causes activation of amino acids and on the other, it helps binding of amino acids to the tRNA end. In the cell, for different amino acids, there are different aminoacyl tRNA synthetases. The enzyme also shows specificity to the tRNA. Therefore, for translation, more than one type of aminoacyl tRNA synthetase is required to be present.
- Peptidyl Transferase: On the large ribosomal sub-unit, this enzyme is present. It helps in the formation of peptide bonds between two amino acids.
- Transformylase: This enzyme converts the initial methionine into formylated methionine in prokaryotic cells.
4. Protein Factors at different steps of protein Synthesis:
Various protein factors regulate the process of translation. In E. coli cells at the initiation phase three initiation factors namely IF1, IF2, and IF3 promote the formation of the initiation complex. During the elongation phase three more factors namely EF Tu, EF Ts, and EF G are involved. In the last phase (Termination) again two factors namely RF1 and RF2 are needed to stop the synthesis of the polypeptide chain.
5. Energy Donors:
In the process of translation, energy is principally donated either by ATP or by GTP. Actually, ATP is needed at the stage of activation of amino acids. In the other stages, GTP is the primary energy donor.
Polypeptide chain synthesis in the cell occurs over the ribosome in the cell cytoplasm. The ribosome is considered a non-specific workbench for amino acid polymerization. Two subunits of ribosome participate in the process by different capacities, though ultimately they work together.
In prokaryotes, the ribosome is 70S type and this is formed by two subunits as 30S and 50S. On the other hand in eukaryotes, the ribosomes are 80S type with sub-units as 40S and 60S. The complete ribosome is required for translation to be continued, though in the process of initiation, the small ribosomal sub-unit (30S in the case of prokaryotes and 40S in the case of eukaryotes) only initiates the process. The ribosome acts as a non-specific work bench for polypeptide chain synthesis.
The function of tRNA is to carry the amino acid to the site of polymerization. However, for the transportation of different amino acids, there are different tRNA species in the cell.
Mechanism of Translation
In the process of translation on the whole, a number of amino acids are polymerized under the direction of mRNA codons over the ribosomal surface. Throughout the whole process of such polypeptide formation, a number of events occur sequentially and they may be grouped into three phases namely initiation, elongation, and termination. The tRNA molecules carry the amino acids on the ribosome surface, where they are polymerized one by one. However, the amino acids that are transported by tRNA are processed by a mechanism called acylation. This acylation occurs through a two step-activation of amino acid and charging of tRNA. In the activation process, the amino acid binds with one ATP molecule and in the charging phase, the amino acids bind with the tRNA. Hence, prior to discussion of the steps of translation, activation of amino acids and charging of tRNA should be discussed.
(a) Activation of Amino Acids:
At this step one amino acid may react with one ATP molecule under the influence of the enzyme (specific) aminoacyl tRNA synthetase and an amino acid-AMP-enzyme complex is formed. The overall reaction may be shown in the following way.
Amino Acid + ATP → Amino acid-AMP-Enzyme + ppi
(b) Charging of tRNA:
Following the activation of amino acid, the amino acid-AMP-enzyme complex reacts with a specific tRNA (as the enzyme has specificity to a tRNA). In this reaction step, the tRNA molecule binds with the specific site of the enzyme. The amino acid may then bind with the 3′ OH end of the tRNA under the influence of the same enzyme. As a result, amino acyl tRNA is formed and both the enzyme and AMP are released.
Amino Acid-AMP-enzyme + tRNA → Amino acyl tRNA + Enzyme + AMP
(c) Formylation of Methionine:
The first amino acid incorporated during the initiation of translation is always methionine and this methionine is transported by a special type of methionine-specific tRNA. In the case of prokaryotic cells, this tRNA is called tRNA-fMet because the methionine it carries is always methylated. The tRNAfMet first binds with methionine and then the charged tRNA-fMet, is attacked by transformylase when methionine attached to the tRNA end is formylated, i.e., a formyl group is attached to the methionine. The three basic stages of protein synthesis are Initiation, Elongation, and Termination.
1. Initiation Phase:
Before initiation of translation, two ribosomal sub-units are separated from each other. In E. coli, 70S ribosomes are separated as 30S and 50S sub-unit. When IF3 binds with the 30S subunit it gets separated from the 50S subunit. Then IF1 comes to bind with it. At this stage mRNA may bind with the ribosome at a sequence (Shine Dalgarno) along its 5′ end. As a result of this, a complex formed of 30S-IF3-IF1-mRNA is formed.
The initiation codon AUG now comes over the 30S sub-unit. After this, the f-Met tRNAf-Met enters the ribosome complex and the charged tRNA sits on the AUG codon with its anticodon. Entry of fMet tRNAf-Met is promoted by IF2 and a GTP molecule. On completion of the incorporation of f-Met tRNAf-Met on the ribosome (the 30S) and mRNA moiety, the initiation factors, IF3, IF1, and IF2 are released from the 30S initiation complex when GTP is hydrolyzed into GDP and Pi. At this stage, the large ribosomal subunit (50S) joins the 30S-mRNA charged tRNA moiety to form a 70S initiation complex. This marks the completion of initiation.
2. Elongation Phase:
With the formation of the 70S initiation complex three sites are developed on the ribosome namely A site (Amino acyl site), P site (Peptidyl site), and E site (Exit site). At this stage, the initiation codon and the initial tRNA with its formylated methionine occupy the P site, while the A site carries the second codon next to AUG on the mRNA.
Therefore, the A site becomes accessible to the second tRNA carrying the codon-specific amino acid. The second specific aminoacyl tRNA then enters the A site of the ribosome and the reaction is promoted by elongation factor EF-Tu and GTP. EF-Tu and GTP first bind with the aminoacyl tRNA. GTP is hydrolysed into GDP and Pi. The elongation factor EF-Ts then joins with the EF-Tu removing the GDP attached to it. As both the P and A sites of the ribosome remain occupied by the aminoacyl tRNA, the first polymerization reaction may occur under the influence of peptidyl transferase. The attached -COOH end of the formylated methionine at the P site reacts with the free NH2 end of the second amino acid attached to the tRNA end at the A site and as a result, a peptide bond (—CO.NH—) is formed between two amino acids. With this reaction, tRNA at the A site becomes attached to a di-peptide, and tRNA at the P site becomes free from amino acid.
Following this, a reaction occurs when the dipeptidyl tRNA at the A site along with the codon of mRNA is transferred from the A site to the P site of the ribosome. As a result of this, the third codon (3rd from AUG) of the mRNA comes to the A site. Translocation reaction is carried out by EF-G and GTP. In this exercise, a relative movement of ribosome and mRNA may be noticed, when the mRNA codon moves from the A site to the P site. After the translocation reaction is over, both EF-G and GTP are dissociated from the ribo¬some. GTP is hydrolyzed in this reaction to form GDP and Pi. This reaction promotes the movement of tRNA from the P site to the E site for its dislocation from the ribosome and tRNA of the A site is moved to the P site.
As the A site of the ribosome becomes occupied by a third codon, it becomes accessible to another specific aminoacyl tRNA. The compatible aminoacyl tRNA gets entry with the action of EF-Tu and GTP again. The specific aminoacyl tRNA enters the A site. It sits on the specific codon at the A site and following this, EF-Tu and GTP in hydrolyzed form become dissociated from the ribosome. Then the second polymerization reaction may occur under the influence of peptidyl transferase. In this reaction the attached -COOH end of the dipeptide at the P site reacts with free -NH2 of the amino acid at the A site to form a peptide bond, so that a tripeptide is formed which remains attached to the tRNA at the A site. With this exercise, the tRNA at the P site becomes free from amino acids. Then again a translocation reaction may be accomplished by EF-G and a GTP molecule.
In this reaction, the tripeptidyl tRNA is transferred from the A site to the P site and the tRNA of the P site to the E site for its dislocation, whereby the fourth codon of mRNA comes to the A site takes its position so that this site becomes accessible to another specific and compatible aminoacyl tRNA. In this way, polymerization and translocation reactions continue cyclically one after another following entry of aminoacyl tRNA to the A site of the ribosome in a sequential fashion until a termination codon (UAG/UGA/UAA) reaches the A site. By this time a quite long chain of amino acids may be formed on the ribosome. However, the number of amino acids in a polypeptide chain depends on the number of codons present in the mRNA.
3. Termination Phase:
As the termination codon reaches the A site of the ribosome further incorporation of aminoacyl tRNA to the A site becomes impossible and then termination of polypeptide chain synthesis occurs. At this phase activity of two termination factors may be observed. These factors are known as RF1 and RF2. RF1 and RF2 recognize the termination codon at the A site and also are capable of releasing the polypeptide chain from tRNA at the P site of the ribosome. Out of three termination codons, UAG and UAA may be recognized by RF1, while UGA or UAA may be recognized by RF2. RF1 or RF2 first binds with the termination codon at the A site and influences peptidyl transferase. As a consequence polypeptide chain gets detached from the tRNA end and binds with a molecule of water. tRNA then being free from the amino acid chain is dissociated from the ribosome. The subunit of ribosome i.e., 30S and 50S may be dissociated again to carry out translation of another molecule of mRNA. Involvement of another termination factor known as RF3 has been reported and this is combined with GTP to stimulate RF1 or RF2 binding.
Initiation Factors of Prokaryotes:
|IF1||Stabilizes initiation complex and helps IF2 & IF3 to function properly.|
|IF3||Binds to the 30S subunit of the ribosome and promotes 50S to be dissociated.|
|IF2||Promotes incorporation of tRNA.Met to bind with 30S subunit.|
Some Differences between Prokaryotic & Eukaryotic Transcription:
|σ-factor present.||σ-factor absent.|
|The promoter region contains a print-now box at -10 positions. TATA box and CAT box are absent.||The promoter region contains; a TATA box located 35 to 25 upstream; a CAT box located ~70 nucleotides upstream; GG box located ~110 nucleotides upstream. The pribnow box is absent in eukaryotes.|
Translation in Eukaryotes
Translation in eukaryotes resembles that observed in prokaryotes. However, some basic differences in the machinery of translation may be observed in eukaryotes. There are 80S ribosomes, over ten initiation factors, only two elongation factors, two termination factors, and monocistronic mRNA, to accomplish translation in eukaryotes.
(a) Eukaryotic Initiation:
About nine initiation factors promote initiation in eukaryotic cells. In contrast to the prokaryotic initiation factor, the functions of four initiation factors are typical for the eukaryotic system.
Initiation Factors of Eukaryotes:
|elF2||binding of mettRNAmet with mRNA|
|First factors to be combined with the 40S and initiate the process|
|CBPI||Binds with the cap of mRNA|
|Being associated with mRNA facilitate locating AUG on mRNA|
|elF5||Facilitates binding of 60S subunit by dissociating other factors from 40S|
In eukaryotes prior to binding of mRNA with the ribosome, initial tRNA carrying methionine binds with the 40S subunit. But this binding is facilitated by eIF2. The initial methionine incorporated is not formylated in this case and to carry this methionine a specific tRNA known as tRNAi is required. However, eIF3 and eIF4C first bind with the 40S subunit and promote subsequent reactions. The eIF2 in association with GTP forms a complex with RNAmet to bind with mRNA. Following this CBP1 (Cap binding protein) binds with the 5′ cap of mRNA which is followed by binding of the factors eIF4A, eIF4B, and eIF4F. These together not only help the binding of mRNA with the complex with the 40S but also help scanning of mRNA for locating initiation codon: AUG in mRNA. In the absence of the Shino-Dalgarno sequence, the AUG codon is embedded in a short sequence called the Kozak sequence.
The 18S rRNA of the 40S subunit of ribosome does not contain any complementary sequence for mRNA binding. In this mRNA binding step, one ATP molecule is used up. With the binding of mRNA with the 40S complex it is converted into a 48S initiation complex. Following this eIF5 gets associated with the initiation complex so that other initiation factors are dissociated. At this stage, the 60S subunit may join the initiation complex to form the 80S initiation complex. It is noteworthy, for the purpose of translation all amino acids are activated in the same fashion as occurs in prokaryotes, and charging of tRNA occurs following the activation of the amino acid.
(b) Elongation in Eukaryotes:
The elongation cycle in this case in closely similar to that of prokaryotes. There are two elongation factors namely EF1 and EF2 that are involved in eukaryotic elongation. EF1 serves the functions of EFTu and EFTs. But EF2 is analogous to the EFG of prokaryotes.
(c) Termination in Eukaryotes:
Termination in eukaryotes may be accomplished by only one factor known as RF which binds with ribosome along with GTP molecule to promote all the activities required for the process of termination of translation.
Energy Expenditure for Translation
It is evident from the use of ATP and GTP utilized at different steps of polypeptide chain formation that the formation of the polypeptide chain is very expensive. During the activation of amino acids two high-energy bonds are utilized from ATP. During incorporation of the aminoacyl tRNA to the ribosomal site one molecule of GTP is hydrolyzed to GDP and Pi. Again at the step of translocation, one GTP is hydrolyzed to GDP and Pi. Therefore, in total at least four high-energy bonds are utilized to form a peptide bond.
Polysomes and Translation
A single mRNA molecule may be associated with a number of ribosomes to form polysomes. Actually being associated with several ribosomes several polypeptide chains may be produced simultaneously within a short period of time. Further, in bacterial cells mRNA lasts only for a few minutes and they may be degraded by nucleases. Therefore, polysome assembly may promote a high rate of polypeptide synthesis with maximum efficiency.
Post Translational Modification
The polypeptide chain which is formed just after translation is required to be processed by the method called post-translational modification. The linear dimensional amino acid chain is converted to a three-dimensional structure of protein when a protein usually becomes biologically active. In both prokaryotic and eukaryotic systems the newly made protein undergoes modification in a number of ways to achieve functional conformation. The different post-translational modifications may be discussed in the following manner.
1. In both prokaryotes and eukaryotes the amino acid at both the amino end and carboxylic end are modified. Formylated methionine at the amino end of the prokaryotic nascent polypeptide is deformylated by the enzyme deformylase. In many cases, the first methionine is enzymatically removed. In eukaryotes, 50% of cases exhibit acetylation of the amino acid at the 5′ end.
2. Some polypeptide chains contain signal sequences at the amino end for directing the protein to its final destination. During processing these sequences are removed by peptidases.
3. In certain cases the hydroxyl group of some amino acid in the polypeptide chain (serine, threonine, tyrosine) is replaced by phosphoric acid by an enzymatic process. Again aspartic acid and glutamic acid may additionally be added to the carboxyl group. In some proteins, lysine is methylated.
4. Polypeptide chain being combined with carbohydrate chain forms glycoprotein.
5. In eukaryotes sometimes proteins become combined with isoprenyl group as in G protein.
6. Both in eukaryotes and prokaryotes some proteins are found to be combined with some prosthetic group for their function as found in cytochrome C and acyl CoA carboxylase.
7. Some proteins are originally synthesized as large molecules and by proteolytic cleavage and digestion they are modified into small molecules at active state. Insulin and trypsin show this type of modification.
8. In some proteins folded polypeptide chain remains in stabilized condition due to the formation of disulfide bonds between the cysteine residues.
9. Besides the above-noted modifications primary protein, i.e., the polypeptide chain, attains alpha helical and beta pleated configuration due to the formation of a maximum number of hydrogen bond formation as well as ionic hydrophobic interactions. Such configurations of primary protein are its secondary structures. The protein in the second configuration is folded further to attain tertiary configuration due to intermolecular interactions. Several proteins like protein disulphide isomerase (PDI), peptidyl-prolyl cis-trans isomerase (PPI ase), and chaperones play a significant roles in (Gelting and Sambrook, 1992) folding of the polypeptide chain. Some proteins again attain a quaternary configuration by further assembly and modification.
Inhibitors of Protein Synthesis:
The majority of known antibiotics may block protein synthesis at various steps. These antibiotics, are produced by certain bacteria and are toxic to bacterial pathogens but harmless to animals. Therefore, such bacterial toxins are medically used to control disease-causing bacteria in man. Several examples of such antibiotics and their role in blocking protein synthesis are listed below.
Some Antibiotics Inhibiting Protein Synthesis in Bacteria:
|Chloramphenicol||Blocks function of peptidyl transferase on large ribosomal subunit.|
|Erythromycin||Translocation is prevented.|
|Puromycin||Causes premature chain termination both in prokaryotes and eukaryotes as it is an aminoacyl tRNA analogue.|
|Streptomycin||Results mRNA misreading and inhibits initiation.|
Difference between Prokaryotic and Eukaryotic Translation:
|Basis of Difference||Prokaryotic translation||Eukaryotic translation|
|Mode of Activity||Translation and transcription are coupled.||Translation and transcription are separate.|
|mRNA||It involves the mRNA that is synthesized in the cytoplasm.||mRNA is synthesized in the nucleus and then transported to the cytoplasm for translation.|
|Cap||It is cap-dependent.||It is usually cap-dependent and also may be cap-independent.|
|Ribosome||The process involves 70S ribosome.||It involves an 80S ribosome.|
|Speed||The process is fast.||The process is slow.|
|Initiation factor||There are 3 initiation factors.||There are 9 initiation factors.|
|Releasing factor||Releasing factors act as RF1 and RF2||Releasing factors act as eRF.|