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Essay # 1. Introduction to Translation of mRNA:
Translation is a process where genetic information is translated from a “nucleic acid language” in to an “amino acid language”. It is catalyzed by ribosome, which contains proteins and ribosomal RNA (rRNA). Translation is a RNA directed synthesis of polypeptides. This process requires all three classes of RNA, mRNA, rRNA and transfer RNA (t-RNA) which can bind to three base pair codons on a messenger RNA (mRNA) and also carry the appropriate amino acid encoded by the codon. The chemistry of peptide bond formation is relatively simple; the processes leading to the formation of a peptide bond are exceedingly complex.
The tRNAs carry activated amino acids into the ribosome which is composed of rRNA and ribosomal proteins. The ribosome is associated with the mRNA ensuring correct access of activated tRNAs and containing the necessary enzymatic activities to catalyze peptide bond formation. The ribosome is inactive when it exists as two subunits (a large one and a small one) before it contacts an mRNA. The small unit of the ribosome initiates the process of translation when it encounters an mRNA in the cytoplasm.
Translation proceeds in an ordered process. First accurate and efficient initiation occurs, and then chain elongation and finally accurate and efficient termination must occur. All these three processes require specific proteins, some of which are ribosome associated and many others are separate from the ribosome, but may be temporarily associated with it. The first A-U-G codon on the 5′ end of the mRNA acts as a “start” signal for the translation machinery and codes for the introduction of a methionine amino acid. Initiation is completed when the methionine tRNA occupies one of the two binding sites on the ribosome.
First site is the site where the growing peptide will reside, it is known as the P site and another site just to the 3′ direction of the P site; it is known as the A site where the incoming tRNA is attached. Every protein begins with the methionine amino acid, not all proteins will ultimately have methionine at one end. If the “start” methionine is not needed, it is removed before the new protein goes to work
The ribosome binds to the mRNA at the start codon (AUG) that is recognized only by the initiator tRNA. The ribosome proceeds to the elongation phase of protein synthesis. During this stage, complexes, composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon. The ribosome moves from codon to codon along the mRNA. Amino acids are added one by one, translated into polypeptidic sequences dictated by DNA and represented by mRNA. At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome.
Activation of Amino Acids:
It is the step in which each of the participating 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 synthetase 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.
The pyrophosphate is hydrolyzed to two inorganic phosphates (2pi). Each tRNA and the amino acid it carries, is recognized by individual aminoacyl-tRNA synthetases. Activation of amino acids requires energy in the form of ATP. First the enzyme attaches the amino acid to the a-phosphate of ATP with the concomitant release of pyrophosphate. This is termed an aminoacyl-adenylate intermediate. Then the enzyme catalyzes transfer of the amino acid to either the 2′- or 3′-OH of the ribose portion of the 3-terminal adenosine residue of the tRNA generating the activated aminoacyl-tRNA, this is the reversible reaction.
Accurate recognition of the correct amino acid as well as the correct tRNA is different for each aminoacyl-tRNA synthetase. Different amino acids have different R groups; the enzyme for each amino acid has a different binding pocket for its specific amino acid. It is absolutely necessary that the discrimination of correct amino acid and correct tRNA be made by a given synthetase prior to release of the aminoacyl-tRNA from the enzyme. After the product is released; there is no means thus proof-read whether a given tRNA is coupled to its corresponding tRNA (Fig. 8.13).
Charging of tRNA:
Here the amino acid AMP-enzyme complex joins with the amino acid binding site of its specific tRNA, where its COOH group bonds with the OH group of the terminal base triplet CCA. The reaction is catalyzed by the same enzyme, aminoacyl tRNA synthetase. The resulting tRNA-amino acid complex is called a charged tRNA. AMP and enzyme are released. The released 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 and form a polypeptide. The tRNA amino acid complex moves to the ribosomes, the site of protein synthesis.
Activation of Ribosome:
It is the step in which the smaller and the larger subunits of ribosome are joined together. TKis is brought about by mRNA chain. The mRNA joins the smaller ribosomal subunit with the help of the first codon by a base pairing with an appropriate sequence on rRNA. The combination of the two is called initiation complex. The larger subunit later joins the small subunit, forming active ribosome. Activation of ribosome by mRNA requires proper concentration of Mg++.
Essay # 2. Types of Translation of mRNA:
A. Prokaryotic Translation of mRNA:
Polypeptide Formation in Prokaryotes:
It involves three events:
(a) Initiation,
(b) Elongation and
(c) Termination of polypeptide chain.
(a) Initiation:
Translation in prokaryotes involves the assembly of the components of the translation system which are the two ribosomal subunits (small and large), the mRNA to be translated, the first (formyl) aminoacyl tRNA (the tRNA charged with the first amino acid), GTP (as a source of energy), and three initiation factors (IF1, IF2, and IF3) which help the assembly of the initiation complex.
The ribosome has three sites- the A site, the P site, and the E site. The A site is the point of entry for the aminoacyl tRNA (except for the first aminoacyl tRNA, fMet-tRNAfMet, which enters at the P site). The P site is where the peptidyl tRNA is formed in the ribosome, while in E site which is the exit site of the now uncharged tRNA after it supplies its amino acid to the growing peptide chain.
The mRNA chain has at its 5′ end an “initiator” or “start” codon (AUG), its recognition that signals the beginning of polypeptide formation (Fig. 8.14). This codon lies close to the P site of the ribosome. In the polycistronic prokaryotic RNAs this AUG codon is located adjacent to a Shine-Delgarno element in the mRNA. The Shine-Delgarno element (Fig. 8.15) is recognized by complimentary sequences in the small subunit rRNA (165 in E. coli).
The amino acid formylmethionine (methionine in eukaryotes) initiates the process. It is carried by tRNA having an anticodon UAC which bonds with the initiator codon AUG of mRNA. Initiation factors (IF1, IF2 and IF3) and GTP promote the initiation process. The large ribosomal subunit now joins the small subunit to complete the ribosome. At this stage, GTP is hydrolysed to GDP. The ribosome has formylmethionine bearing tRNA at the P site. Later, the formylmethionine is changed to normal methionine by the enzyme deformylase in prokaryotes. If not required, methionine is later separated from the polypeptide chain by a proteolytic enzyme aminopeptidase (Fig. 8.16).
In the initiation process of protein synthesis first Initiation factors IF-1 and IF-3 bind to the 305 ribosomal subunit, prevents 30S and 50S subunits combining prematurely. Binding of the mRNA to the 30S subunit takes place in such a way that the initiation codon (AUG) binds to a precise location on the 30S subunit. The prevention of subunit re-association allows the pre-initiation complex to form. Binding of IF-1 helping to insure that the initiation aminoacyl-tRNA, fMet-tRNAfMet can bind only in the P site and other aminoacyl-tRNA can bind in the A site during initiation. The initiation of translation requires recognition of an AUG codon. In the polycistronic prokaryotic RNAs, AUG codon is located adjacent to a Shine-Delgarno element in the mRNA. The Shine-Delgarno element is recognized by complimentary sequences in the small subunit rRNA (165 in E. coli). IF-2 is a small GTP- binding protein binds the initiator fMet-tRNA and helps it to dock with the small ribosome subunit.
(b) Elongation:
Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. The growing protein exits the ribosome through the polypeptide exit tunnel in the large subunit (Fig. 8.17).
Three elongation factors (EF Tu, EF Ts and EF G) assist in the elongation of the polypeptide chain. Elongation starts when the fmet-tRNA enters the P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation factor-Tu (EF-Tu), a small GTPase. Now the P site contains the beginning of the peptide chain of the protein to be encoded and the A site has the next amino acid to be added to the peptide chain. The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site.
This process, known as peptide bond formation, is catalyzed by a ribozyme peptidyltrans- ferase, an activity intrinsic to the 23 S ribosomal RNA in the 50S ribosomal subunit. A charged tRNA molecule along with its amino acid, proline, for example, enters the ribosome at the A site. Its anticodon GGA locates and binds with the complementary codon CCU of mRNA chain by hydrogen bonds. The amino acid methionine is transferred from its tRNA onto the newly arrived proline tRNA complex where the two amino acids join by a peptide bond.
The process is catalyzed by the enzyme peptidyl transferase located on the ribosome. In this process, the linkage between the first amino acid and its tRNA is broken, and the – COOH group now forms a peptide bond with the free -NH2 group of the second amino acid. Thus, the second tRNA carries a dipeptide, formylmethionineproline. The energy required for the formation of a peptide bond comes from the free energy released by separation of amino acid (formylmethionine or methionine) from its tRNA.
The first tRNA, now uncharged, separates from mRNA chain at the P site of the ribosome and returns to the mixed pool of tRNAs in the cytoplasm. Here, it is now available to transport another molecule of its specific amino acid. Now the ribosome moves one codon along the mRNA in the 3′ direction. With this, tRNA dipeptide complex at the A site is pulled to the P site. This process is called translocation. It requires GTP and a translocase protein called EF-G factor. The GTP is hydrolysed to GDP and inorganic phosphate to release energy for the process.
At this stage, a third tRNA molecule with its own specific amino acid, arginine, for example arrives at the A site of the ribosome and binds with the help of anticodon AGA to the complementary codon UCU of the mRNA chain. The dipeptide formylmethionineproline is shifted from the preceding tRNA on the third tRNA where it joins the amino acid arginine again with the help of peptidyl transferase enzyme. The dipeptide, thus, becomes a tripep- tide, formyl-methionine-proline-arginine. The second tRNA being now uncharged, leaves the mRNA chain, vacating the P site. The tRNAtripeptide complex is translocated from A site to P site.
The entire process involving arrival of tRNA-amino acid complex, peptide bond formation and translocation is repeated. As the ribosome moves over the mRNA, all the codons of mRNA arrive at the A site one after another, and the peptide chain grows. Thus, the amino acids are linked up into a polypeptide in a sequence communicated by the DNA through the mRNA. A polypeptide chain which is in the process of synthesis is often called a nascent polypeptide. Since tRNAs are linked to mRNA by codon-anticodon base-pairing, tRNAs move relative to the ribosome taking the nascent polypeptide from the A site to the P site and moving the uncharged tRNA to the E exit site. This process is catalyzed by elongation factor G (EF-G). The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA bind to the A site, until the ribosome reaches a stop codon on mRNA (UAA, UGA, or UAG). The growing polypeptide chain always remains attached to its original ribosome, and is not transferred from one ribosome to another. Only one polypeptide chain can be synthesized at a time on a given ribosome.
(C) Termination:
Termination occurs when one of the three termination codons moves into the A site. These codons are not recognized by any tRNAs. It is not joined by the anticodon of any tRNA amino acid complex. Hence, there can be no further addition of amino acids to the polypeptide chain. Instead, they are recognized by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop codons). These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome.
A third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end of the termination process. The release is catalyzed by the peptidyl transferase enzyme, the same enzyme that forms the peptide bonds. The ribosome jumps off the mRNA chain at the stop codon and dissociates into its two sub- units. The completed polypeptide (amino acid chain) becomes free in the cytoplasm. The ribosomes and the tRNAs on release from the mRNA can function again in the same manner and result in the formation of another polypeptide of the same protein.
B. Eukaryotic Translation of mRNA:
Initiation of translation in both prokaryotes and eukaryotes requires a specific initiator tRNA, tRNAmet, that is used to incorporate the initial methionine residue into all proteins but tRNAmet is specific for initiation in eukaryotes.
Eukaryotic Initiation Factors and their Functions:
The specific non-ribosomally associated proteins required for accurate translational initiation are termed initiation factors. In E. coli they are called Ifs, while in eukaryotes they are denoted as elFs.
Numerous elFs have been identified as shown in Table 8.4:
(a) Initiation:
Initiation of translation requires four basic specific steps- The ribosome must dissociate into its’ 405 and 605 subunits. A ternary complex i.e. the preinitiation complex is formed involving the initiator, GTP, eIF-2 and the 405 subunit. The mRNA is bound to the preinitiation complex. The 605 subunit associates with the preinitiation complex in order to form the 80S initiation complex. The initiation factors eIF-1 and eIF-3 bind to the 405 ribosomal subunit favoring anti-association to the 605 subunit. The prevention of subunit re-association thus allows the pre-initiation complex to form (Fig. 8.18).
The first step in the formation of the pre-initiation complex is the binding of GTP to elF-2 to form a binary complex. eIF-2 is composed of three subunits, α, β and γ. The binary complex then binds to the activated initiator tRNA, i.e. met-tRNAmet forming a ternary complex that then binds to the 40S subunit forming the 43 S pre-initiation complex. The pre-initiation complex is stabilized by an earlier association of eIF-3 and eIF-1 to the 40S subunit. The cap structure of eukaryotic mRNAs is bound by specific elFs prior to association with the pre-initiation complex. Cap binding is accomplished by the initiation factor eIF-4F. This factor is actually a complex of 3 proteins; eIF-4E, A and G. The protein eIF-4E is a 24 kDa protein which physically recognizes and binds to the cap structure. eIF-4A is a 46 kDa protein which binds and hydrolyzes ATP and exhibits RNA helicase activity. Unwinding of mRNA secondary structure is necessary to allow an access of the ribosomal subunits. eIF-4G aids in binding of the mRNA to the 43 S preinitiation complex.
Once the mRNA is properly aligned onto the preinitiation complex and the initiator met- tRNAmet is bound to the initiator AUG codon (a process facilitated by eIF-1) the 60S subunit associates with the complex. The association of the 60S subunit requires the activity of eIF-5 which is already bound to the preinitiation complex. The energy needed to bring about the formation of the 80S initiation complex from the hydrolysis of the GTP bound to eIF-2.
The GDP bound form of eIF-2 then binds to eIF-2B which stimulates the exchange of GTP for GDP on eIF-2. When GTP is exchanged eIF-2B dissociates from eIF-2. This is termed as elF -2 cycle. This cycle is absolutely required for eukaryotic translational initiation to occur. The GTP exchange reaction can be affected by phosphorylation of the a-subunit of eIF-2.
At this stage the initiator met-tRNA is bound to the mRNA within a site of the ribosome P-site. The other site within the ribosome to which incoming charged tRNAs bind is the A- site for amino acid site. The eIF-2 cycle involves the regeneration of GTP-bound eIF-2 following the hydrolysis of GTP during translational initiation. When the 405 preinitiation complex is engaged with the 60S ribosome to form the 80S initiation complex, the GTP bound to eIF-2 is hydrolyzed providing energy for the process of association. In order to undergo additional rounds of translational initiation to occur, the GDP bound to eIF-2 must be exchanged for GTP. This is the function of eIF-2B which is also called guanine nucleotide exchange factor (GEF).
(b) Elongation:
The process of elongation too requires specific non-ribosomal proteins. In E. coli these are EFs and in eukaryotes eEFs. Elongation of polypeptides occurs in a cyclic manner such that at the end of one complete round of amino acid addition the A site is available empty and ready to accept the incoming aminoacyl-tRNA as dictated by the next codon of the mRNA. This means that not only does the incoming amino acid need to be attached to the peptide chain but the ribosome must also move down the mRNA to the next codon.
Each incoming aminoacyl-tRNA is brought to the ribosome by an eEF-la-GTP complex. When the correct tRNA is registered into the A site the GTP is hydrolyzed and the eEF-la-GDP complex dissociates. For additional translocation events the GDP must be exchanged with GTP. This is carried out by eEF-lpy similarly to the GTP exchange that occurs with eIF-2 catalyzed by eIF-2B.
The peptide attached to the tRNA in the P site is transferred to the amino group at the aminoacyl-tRNA in the A site. This reaction is catalyzed by peptidyltransferase. This process is termed transpeptidation. The elongated peptide now resides on a tRNA in the A site.
The A site needs to be free in order to accept the next aminoacyl-tRNA (Fig. 8.19). The process of moving the peptidyl-tRNA from the A site to the P site is termed, translocation. Translocation is catalyzed by eEF-2 coupled to GTP hydrolysis. In the process of translocation the ribosome is moved along the mRNA such that the next codon of the mRNA resides under the A site. Following the translocation eEF-2 is released from the ribosome. The cycle can now begin again. The ability of eEF-2 to carry out translocation is regulated by the state of phosphorylation of the enzyme, when phosphorylated the enzyme is inhibited. Phosphorylation of eEF-2 is catalyzed by the enzyme eEF2 kinase (eEF2K).
(c) Termination:
Similar to initiation and elongation, transiationai termination requires specific protein factors identified as releasing factors, RFs in E. coli and eRFs in eukaryotes. There is one eRFs in eukaryotes. The signals for termination are the same in both prokaryotes and eukaryotes. These signals are termination codons present in the mRNA. There are 3 termination codons, UAG, UAA and UGA.
In E. coli the termination codons UAA and UAG are recognized by RF-1, whereas RF-2 recognizes the termination codons UAA and UGA. The eRF binds to the A site of the ribosome in conjunction with GTP. The binding of eRF to the ribosome stimulates the peptidy- transferase activity to transfer the peptidyl group to water instead of an aminoacyl-tRNA. The resulting uncharged tRNA left in the P site is expelled with concomitant hydrolysis of GTP. The inactive ribosome then releases its mRNA and the 80S complex dissociates into the 40S and 60S subunits ready for another round of translation.