Prokaryotic Translation (Protein Synthesis)
- Translation involves translating the sequence of a messenger RNA (mRNA) molecule to a sequence of amino acids during protein synthesis.
- It is the process in which ribosomes in the cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA.
- Ribosomes exist normally as separate subunits that are composed of protein and rRNA.
- The subunits come together to form a ribosome when they bind to an mRNA, near its 5’ end.
- On binding to an mRNA, the ribosome reads the nucleotide sequence from the 5’ to 3’ direction, synthesizing the corresponding protein from amino acids in an N-terminal (amino-terminal) to C-terminal (carboxyl terminal) direction.
- Ribosomes are located in the cytosol, either freely floating or associated with the endoplasmic reticulum.
- They serve to synthesize proteins.
Ribosomal Sites for Protein Translation
Each prokaryotic ribosome, shown schematically, has three binding sites for tRNAs.
- The aminoacyl-tRNA binding site (or A site) is where, during elongation, the incoming aminoacyl-tRNA binds.
- The peptidyl-tRNA binding site (or P site) is where the tRNA linked to the growing polypeptide chain is bound.
- The exit site (or E site) is a binding site for tRNA following its role in translation and prior to its release from the ribosome.
All three sites (A, P and E) are formed by the rRNA molecules in the ribosome.
THE PROCESS OF TRANSLATION
Protein synthesis (or translation) takes place in three stages:
- Elongation and
- During initiation, the mRNA–ribosome complex is formed and the first codon (always AUG) binds the first aminoacyltRNA (called initiator tRNA).
- During the elongation phase, the other codons are read sequentially and the polypeptide grows by addition of amino acids to its C-terminal end.
- This process continues until a termination codon (Stop codon), which does not have a corresponding aminoacyl-tRNA with which to base pair, is reached.
- At this point, protein synthesis ceases (termination phase) and the finished polypeptide is released from the ribosome.
Synthesis of aminoacyl-tRNA
- Synthesis of aminoacyl-tRNAs is crucially important for two reasons:
- Each amino acid must be covalently linked to a tRNA molecule in order to take part in protein synthesis, which depends upon the ‘adaptor’ function of tRNA to ensure that the correct amino acids are incorporated.
- The covalent bond that is formed between the amino acid and the tRNA is a high energy bond that enables the amino acid to react with the end of the growing polypeptide chain to form a new peptide bond.
For this reason, the synthesis of aminoacyl-tRNA is also referred to as amino acid activation.
- Each tRNA molecule has a cloverleaf secondary structure with the anticodon accessible at the end of the anticodon stem loop.
- During synthesis of the aminoacyl-tRNA, the amino acid is covalently bound to the A residue of the CCA sequence at the 3’ end.
- Each tRNA molecule carries only a single amino acid.
- The attachment of an amino acid to a tRNA is catalyzed by an enzyme called aminoacyl-tRNA synthetase.
- A separate aminoacyl-tRNA synthetase exists for every amino acid, making 20 synthetases in total.
The synthesis reaction occurs in two steps.
- The first step is the reaction of an amino acid and ATP to form an aminoacyl-adenylate (also known as aminoacyl-AMP).
- In the second step, without leaving the enzyme, the aminoacyl group of aminoacyl-AMP is transferred to the 3’ end of the tRNA molecule to form aminoacyl-tRNA
The overall reaction is:
Amino acid + ATP + tRNA → aminoacyl-tRNA + AMP + PPi
Initiation of Protein Synthesis
- The first codon translated in all mRNAs is the start codon or initiation codon, AUG which codes for methionine.
- Two different tRNAs are used for the two types of AUG codon; tRNAfMet is used for the initiation codon and is called the initiator tRNA whereas tRNAm Met is used for internal AUG codons.
- In prokaryotes the first amino acid of a new protein is N-formylmethionine (abbreviated fMet). Hence the aminoacyl-tRNA used in initiation is fMet-tRNAfMet.
- A short sequence rich in purines (5’-AGGAGGU-3’), called the Shine–Dalgarno sequence, lies 5’ to the AUG initiation codon and is complementary to part of the 16S rRNA in the small ribosomal subunit.
- Therefore this is the binding site for the 30S ribosomal subunit which then migrates in a 3’ direction along the mRNA until it encounters the AUG initiation codon.
- Initiation of protein synthesis requires proteins called initiation factors (IFs).
- In prokaryotes, three initiation factors (IF-1, IF-2 and IF-3) are essential.
- Because of the complexity of the process, the exact order of binding of IF-1, IF-2, IF-3, fMet-tRNAf is controversial.
- Initiation begins with the binding of IF-1 and IF-3 to the small (30S) ribosomal subunit.
- Their role is to stop the 30S subunit binding to the 50S subunit in the absence of mRNA and fMet-tRNAf Met which would result in a nonfunctional ribosome.
- The small subunit then binds to the mRNA via the Shine–Dalgarno sequence and moves 3’ along the mRNA until it locates the AUG initiation codon.
- The initiator tRNA charged with N-formylmethionine and in a complex with IF-2 and GTP (fMet-tRNAfMet/IF-2/GTP) now binds.
- IF-3 is released.
- The complex of mRNA, fMet-tRNAf Met, IF-1, IF-2 and the 30S ribosomal subunit is called the 30S initiation complex.
- The large (50S) ribosomal subunit now binds, with the release of IF-1 and IF-2 and hydrolysis of GTP, to form a 70S initiation complex.
Elongation of Protein Synthesis
- At the start of the first round of elongation, the initiation codon (AUG) is positioned in the P site with fMet-tRNAfMet bound to it via codon–anticodon base pairing.
- The next codon in the mRNA is positioned in the A site.
- Elongation of the polypeptide chain occurs in three steps called the elongation cycle, namely aminoacyl-tRNA binding, peptide bond formation and translocation:
- The corresponding aminoacyl-tRNA for the second codon binds to the A site via codon–anticodon interaction.
- Binding of the aminoacyl-tRNA requires elongation factor EF-Tu and GTP which bind as an aminoacyl-tRNA/EF-Tu/GTP complex.
- Following binding, the GTP is hydrolyzed and the EF-Tu is released, now bound to GDP.
- Before the EF-Tu molecule can catalyze the binding of another charged tRNA to the ribosome, it must be regenerated by a process involving another elongation factor, EF-Ts.
This regeneration is called the EF-Tu–EF-Ts exchange cycle.
- First, EF-Ts binds to EF-Tu and displaces the GDP. Then GTP binds to the EF-Tu and displaces EF-Ts. The EF-Tu-GTP is now ready to take part in another round of elongation.
Peptide bond formation
- The second step, peptide bond formation, is catalyzed by peptidyl transferase.
- In this reaction the carboxyl end of the amino acid bound to the tRNA in the P site is uncoupled from the tRNA and becomes joined by a peptide bond to the amino group of the amino acid linked to the tRNA in the A site.
- In the third step, a complex of elongation factor EF-G (also called translocase) and GTP (i.e. EF-G/GTP) binds to the ribosome.
- Three concerted movements now occur, collectively called translocation:
- the deacylated tRNA moves from the P site to the E site
- the dipeptidyl-tRNA in the A site moves to the P site, and
- the ribosome moves along the mRNA (5’ to 3’) by three nucleotides to place the next codon in the A site.
- During the translocation events, GTP is hydrolyzed to GDP and inorganic phosphate, and EF-G is released ready to bind more GTP for another round of elongation.
- After translocation, the A site is empty and ready to receive the next aminoacyltRNA.
- The A site and the E site cannot be occupied simultaneously. Thus the deacylated tRNA is released from the E site before the next aminoacyl-tRNA binds to the A site to start a new round of elongation.
- Elongation continues, adding one amino acid to the C-terminal end of the growing polypeptide for each codon that is read, with the peptidyl-tRNA moving back and forth from the P site to the A site as it grows.
Termination of Protein Synthesis
- Eventually, one of three termination codons (also called Stop codons) becomes positioned in the A site. These are UAG, UAA and UGA.
- Unlike other codons, prokaryotic cells do not contain aminoacyl-tRNAs complementary to
- Stop codons. Instead, one of two release factors (RF-1 and RF-2) binds instead.
- RF-1 recognizes UAA and UAG whereas RF-2 recognizes UAA and UGA. A third release factor, RF-3, is also needed to assist RF-1 or RF-2 interaction with the ribosome. Thus either RF-1 + RF-3 or RF-2 + RF-3 bind depending on the exact termination codon in the A site.
- RF-1 (or RF-2) binds at or near the A site whereas RF-3/GTP binds elsewhere on the ribosome.
- The release factors cause the peptidyl transferase activity to transfer the polypeptide to a water molecule instead of to aminoacyl-tRNA, effectively cleaving the bond between the polypeptide and tRNA in the P site.
The free polypeptide now leaves the ribosome, followed by the mRNA and free tRNA, and the ribosome dissociates into 30S and 50S subunits ready to start translation again.
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