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TEXT Translation is simply the decoding of nucleotide sequences on mRNA into the amino acid sequence of a polypeptide. The process of Translation ( synthesis of a polypeptide chain) can be divided into three rather distinct activities: a) Initiationwhich involves binding of ribosomal subunits with mRNA accompanied with tRNA charged with methionine; b) Elongation- in which molecules successive aminoacylated tRNA are brought sequentially on the ribosomes, i.e translating the mRNA molecule and the formation of peptide bond between the successive amino acids, and c) Termination-where the ribosomal units dissociate into individual units and the synthesized polypeptide chain is released. Proteins are the work horses of the cell, controlling virtually every reaction within, as well as providing structure, and serving as signals to other cells. They are long chains of amino acids, and the exact sequence of the amino acids determines the final structure and function of the protein. Instructions for that sequence are encoded in genes (DNA).The synthesis of every protein molecule in a cell is directed by an mRNA, originally copied from DNA. Synthesis of RNA from DNA template is called transcription and the process is catalyzed by an enzyme called RNA polymerase. Next step is the synthesis of polypeptide (protein) from mRNA. Synthesis of a polypeptide from mRNA includes two types of processes: (1) information-transfer process in which the mRNA base sequence determines an amino acid sequence, and (2) chemical process in which the amino acids are linked together. The complete series of events is called Translation. Translation may also be defined as the process by which amino acid sequence of polypeptide is synthesized on a ribosomal complex according to the nucleotide sequence of an mRNA molecule (Fig. 1). Fig.1. Flow of genetic information from DNA to proteins Before discussing the process of Translation, let us describe some ingredients necessary for Translation. The main ingredients of Translation are as under: Messenger RNA: Messenger RNA (mRNA) is an RNA molecule that serves as a template for protein synthesis. It is needed to bring the ribosomal subunits together and to provide the coding sequence of bases that determines the amino acid sequence in the resulting polypeptide chain. Ribosomes: Ribosomes are the sites of protein synthesis ( Translation) in both prokaryotes and eukaryotes. They move along an mRNA molecule and expose codons for appropriate aminoacyl tRNA molecules as per genetic code (Fig. 2b). The amino acids are added to the growing polypeptide chain one by one and the peptide bond formation is catalyzed by rRNA of small subunit of ribosome. In Prokaryotes, Translation occurs on 70S ribosomes whereas in Eukaryotes it occurs on 80S ribosomes (except in mitochondria and chloroplasts) The general structure of prokaryotic and eukaryotic ribosomes is similar, although they differ in size (Fig. 2a). The small subunit (designated as 30S) of prokaryotes consists of the 16S rRNA and 21 proteins; the large subunit (50S) is composed of the 23S and 5S rRNA and 34 proteins. The subunits of eukaryotic ribosomes are larger and contain more proteins than their Eukaryotic counterparts have. The small subunit (40S) of Eukaryotic ribosomes is composed of 18s rRNA and approximately 30 proteins; the larger subunit (60S) contain the 28S, 5.8S and 5S rRNA and about 45 proteins. Transfer RNA ( tRNA): The sequence of amino acids in a polypeptide is determined by the 70S 80S Fig. 2. a-‐Structure of prokaryotic (70S) and eukaryotic (80S) ribosomes; b-‐Genetic code showing t aminoacids base sequence in the mRNA by means of a set of adaptor molecules known as tRNA . The tRNA binds to the mRNA codons (group of three adjacent bases on mRNA) through anticodon site, which contains three complementary bases to codons, and thus bind amino acids adjacent to one another for the formation of peptide bond. The tRNA’s are approximately 70-80 nucleotides long, and have characteristic cloverleaf-like structure (Fig. 3a) that results from complementary base-pairing between different regions of the molecule. All the tRNA molecules have sequence CCA at their 3' terminus (amino acids are covalently attached to the ribose of the terminal A of CCA), the anticodon loop at the other end, one DHU loop, a pseudouridine loop (PψC), and an extra arm or variable loop of 7-10 nucleotides. X ray diffraction analysis has shown that the tRNA’s are made up of two double helices arranged in the shape of an ‘L’ (Fig. 3b). Aminoacids: The pool of 20 amino acids is used in the synthesis of polypeptides or proteins; these are shown in Fig. 2b. Aminoacyl tRNA synthetases: This set of enzymes catalyses the attachment of each amino acid to its corresponding tRNA molecule. A tRNA molecule attached to its amino acid is called an aminoacylated tRNA or charged tRNA. The formation of charged tRNA occurs in two steps: I. Activation of amino acids: This reaction is brought about by binding of an amino acid with the ATP, and is mediated by specific amino acid tRNAsynthetases, as a result of this a complex, known as aminoacyl-AMP enzyme complex, is formed. Amino acid + ATP Aminoacid-AMP enzyme complex +PPi II. Charging of an amino acid on tRNA: The aminoacid-AMP enzyme complex reacts with a tRNA and transfers the amino acid to tRNA, accompanied with the release of AMP and the enzyme. The reaction is highly specific. Aminoacid-AMP enzyme complex + tRNA Enzyme. Charged tRNA + AMP + a b Fig. 3. a-‐ Two dimensional structure of tRNA; b-‐ Comparison of two and three dim of tRNA The synthesis of a polypeptide, i,e. translation is a continues process; but for the sake of simplicity, it is divided into three distinct steps, namely 1. Initiation, 2. Elongation, and 3. Termination. 1. Initiation: The main feature of the initiation is the binding of an mRNA to the small subunit of ribosome, tRNA charged with formylated metionine, followed by the large ribosomal subunit to form the initiation complex. The ribosomes have three sites for tRNA molecules; these are: E (exit) site, from which the uncharged tRNA leave during elongation, P (peptidyl) site, and an A (aminoacyl) site. The initiation proceeds with the binding of the 30S ribosomal subunit with the initiation factors, IF-1 & IF-3. The IF-3 prevents the 30S & 50S sub units from combining prematurely. Factor IF-1 binds at the A site during initiation. The mRNA then binds to the 30S subunit in such a way that the initiating (5')AUG codon is positioned at the P site of ribosome. Bacterial mRNA posses a specific sequence of nucleotides (called the Shine-Dalgarno sequence, named after its discoverers) that resides 5-10 nucleotides before the initiation codon. The Shine-Dalgarno sequence is complementary to a sequence of nucleotides near the 3' end of the 16s rRNA of small ribosomal subunit. It is pairing between complementary bases of Shine-Dalgarno sequence and 16S rRNA, that positions AUG codon at P site of ribosome. The initiation complex consisting of the 30S ribosomal subunit , IF-3, IF-1, and mRNA is joined by both GTP bound IF-2 and the initiating tRNA-fmet. The anticodon of this tRNA pairs with the initiation codon of mRNA. It should be noted that only the first charged tRNA binds with the P site, all the other successively added tRNA attaches at A site of ribosomes. This is followed by joining of 50S ribosomal subunit, but at this step GTP bound to IF-2 is hydrolyzed to GDP and PPi, which are released from the complex. All the three initiation factors depart from the ribosome at this point. At the completion of this process, a functional 70s ribosome, called the initiation complex is formed (Fig. 4). Initiation in eukaryotes proceeds in the similar way as in prokaryotes, i,e. binding of small subunit (40S) with the mRNA followed by charged tRNA and then larger subunit (60S) to form the initiation complex; but initiation in eukaryotes requires atleast 10 initiation factors, designated as eIFs ( Eukaryotic initiation factors), which makes initiation in eukaryotes more complex. Fig. 4. Formation of initiation complex at the expense of hydrolysis of ATP 2. Elongation: After the initiation complex has been formed, translation proceeds by elongation of the polypeptide chain. The ribosome has three sites for tRNA binding, designated the P site (Peptidyl), A site ( Aminoacyl), and E (Exit) site as mentioned earlier. The initiator methionyl tRNA is bound at the P site. The first step in elongation is binding of the next aminoacyl tRNA to the A site by pairing with the second codon of the mRNA. The aminoacyl tRNA is escorted to the A site of the ribosome by an elongation factor EF-TU in prokaryotes, which is complexed with GTP. The GTP is hydrolyzed to GDP as the correct aminoacyl tRNA is inserted into the A site of the ribosome, and the elongation factor bound to GDP is released. The hydrolysis of GTP in elongation is the rate-limiting step, and provides the time interval during which an incorrect aminoacyl tRNA (which would bind less strongly to the mRNA codon) can dissociate from the ribosome, rather than being used in the formation of a polypeptide. The expenditure of a high energy GTP at this step has an important contribution to accurate protein synthesis. It allows time for proof reading of the codon-anticodon pairing before the peptide bond forms. Fig. 5. Eongation (the binding of the second aminoacyl-tRNA and formation of a polypeptide bond) Once EF-TU has left the ribosome, a peptide bond is formed between the amino acids attached with the tRNA on A site and P site (Fig. 5.1). This reaction is catalyzed by the large ribosomal subunit, with RNA playing a critical role; the result is the transfer of the first amino acid at P site to the aminoacyl tRNA at the A site of the ribosome, forming a dipeptide tRNA at this position, and leaving the uncharged initiator tRNA at the P site. The next step in elongation is translocation, which requires another elongation factor known as translocase (or EF-G in prokaryotes), and is again coupled to GTP hydrolysis. During translocation, the ribosome’s move three nucleotides along the mRNA in 5' → 3' direction at each step, positioning the next codon in an empty A site. The binding of a new aminoacyl tRNA to the A site then induces the release of the uncharged (deacylated) tRNA from the E site into the cytosol (Fig. 5.2). At each step of translocation, newer and newer aminoacyl tRNA attaches at the A site, and uncharged tRNA are exited through the E site, as the ribosome moves from codon to codon along the mRNA towards the 3' end. As elongation continues, the EF-TU that is released from the ribosome bound to GDP, must be reconverted to its GTP form. This conversion requires a third elongation factor, EF-Ts, which binds to EF-TU/GDP complex, and promotes the exchange of bound GDP for GTP. This exchange results in the regeneration of EF-TU/GTP, which is now ready to escort a new aminoacyl tRNA in the A site of the ribosome, beginning a new cycle of elongation. Fig 5.2 Elongation (the ribosomes moves one codon towords the 3' end of mRNA translocation) The elongation cycle in eukaryotes is quite similar to that of prokaryotes. The three elongation factors eEF-1α, eEF-1βγ and eEF-2 in eukaryotes are analogous to that of prokaryotic elongation factors EF-TU, EF-TS and EF-G, respectively. Further eukaryotic ribosomes do not have E site, so uncharged tRNA are expelled directly from the P site. 3. Termination: Elongation polypeptide codon, continues also (UAA, called UAG until a nonsense or UGA), of a stop codon is translocated into the A site of the ribosome . Cells do not contain tRNA with anticodon complimentary to these terminating signals , have release recognize instead factors that signals and the they terminate protein synthesis (Fig The release factors 6). have domains thought to mimic the structure of tRNA. Prokaryotic cells contain two release factors that recognize codon; RF-1 recognizes UAA or UAG, and RF- 2 recognizes termination UAA or UGA. In eukaryotes, a single releasing factor eRF-1 recognizes all the three termination codons. The release factors bind to the termination at the A hydrolysis site of and stimulate bond between and polypeptide chain at the P resulting in the release codon tRNA site , of polypeptide from the ribosome. Both prokaryotic and eukaryotic cells also Fig. 6 The termination of protein synthesis in response to a termination codon. contain release factors, RF-3 and eRF-3, respectively, that don’t recognize specific termination codon, but act together with RF-1 (eRF-1), and RF-2 and are thought to dissociate or release the ribosomal units. In most of the cells, single mRNA can be translated simultaneously by several ribosomes in both prokaryotic and eukaryotic ones (particularly in metabolically active cells). As one ribosome moves from the initiation site, another one can bind to the mRNA and begins synthesis of a new polypeptide chain, such a condition is known as polysome or polyribosome. This type of translation helps the cell to amplify the specific protein and meet the excessive demand of the protein within or outside the cell. Fig. 7. Polysome (where more than one ribosome translate same mRNA simultaneously)