protein synthesis biochem.ppt
- 1. PROTEIN BIOSYNTHESIS AND INHIBITION Dr Rina Das Mehta Professor
- 2. Protein Biosynthesis:- The biosynthesis of protein or polypeptide in a living cell is refer to as translation. •Site of protein biosynthesis:- The ribosome is the cellular component on which various functional entities interact to synthesis the protein molecule.The ribosome have different sub-units like 40s and 60s depending on sedimentation rate. mRNA has no affinity for amino acid. Thus, the translation of genetic information in the mRNA nucleotide sequence into amino acid sequence of a protein requires and intermediate adapter molecule. The adapter molecule are tRNA, translating the codons into amino acid sequence.
- 3. 1. Amino acid is the starting material for these biosynthesis . 2. The ribosome is the working bench.They are large complexes which have two dissimilar sub-units i.e. large and small. A eukaryotic ribosome particle is 80S molecule with two subunits of 60S and 40S.The prokaryotic ribosomes are 70s molecules with 2 sub- •Requirements for protein biosynthesis: Protein biosynthesis requires mRNA , tRNA ribosome , amino acids, several proteins factors and ATP as the source of energy.
- 6. THE STEPS INVOLVED IN PROTEIN SYNTHESIS ARE :- 1. Activation of amino acid 2. Protein synthesis proper
- 7. ACTIVATION OF AMINO ACID Amino acid activation (also known as aminoacylation or tRNA charging) refers to the attachment of an amino acid to its Transfer RNA (tRNA). Aminoacyl transferase binds Adenosine triphosphate (ATP) to amino acid, PP is released. Aminoacyl tRNA synthetase binds AMP-amino acid
- 8. During amino acid activation the amino acids (aa) are attached to their corresponding tRNA. The coupling reactions are catalysed by a group of enzymes called aminoacyl-tRNA synthetases (named after the reaction product aminoacyl-tRNA or aa-tRNA). The coupling reaction proceeds in two steps: aa + ATP ⟶ aa-AMP + PP, (pyrophosphate) aa-AMP + tRNA ⟶ aa-tRNA + AMP
- 9. PROTEIN SYNTHESIS PROPER • The protein or polypeptide synthesis occurs on the ribosomes (rather polyribosomes). The mRNA is read in the direction and the polypeptide synthesis proceeds from N-terminal end to C-terminal end. Translation proper is divided into three stages:- • Initiation • Elongation • Termination
- 10. INITIATION OF TRANSLATION • The initiation of translation in eukaryotes is complex, involving at least ten eukaryotic initiation factors (elFs). Some of the elFs contain multiple (3-8) subunits. The process of translation initiation can be divided into four steps: 1. Ribosomal dissociation. 2. Formation of 43S pre-initiation complex. 3. Formation of 48S initiation complex. 4. Formation of 80S initiation complex.
- 11. Ribosomal dissociation: The 80S ribosome dissociates to form 40S and 60S subunits. Two initiating factors namely elF-3 and elF-1A bind to the newly formed 40S subunit, and thereby block its re-association with 60S subunit. For this reason, some workers name elF-3 as anti- association factor.
- 12. Formation of 43S pre-initiation complex: A ternary complex containing met-tRNA’ and elF-2 bound to GTP attaches to 40S ribosomal subunit to form 43S pre-initiation complex. The presence of elF-3 and elF-1 A stabilizes this complex (Note; Met-tRNA is specifically involved in binding to the initiation condon AUGs; hence the superscrip’ is used in met-tRNA’).
- 13. Formation of 48S initiation complex: The binding of mRNA to 43S pre-initiation complex results in the formation of 48S initiation complex through the intermediate 43S initiation complex. This, however, involves certain interactions between some of the elFs and activation of mRNA.
- 14. Formation of 80S initiation complex: 48S initiation complex binds to 60S ribosomal subunit to form 80S initiation complex. The binding involves the hydrolysis of GTP (bound to elF-2). This step is facilitated by the involvement of elF-5. As the 80S complex is formed, the initiation factors bound to 48S initiation complex are released, and recycled. The activation of elF-2 requires elF-2B (also called as guanine nucleotide exchange factor) and GTP. The activated elF-2 (i.e. bound to GTP) requires elF-2C to form the ternary complex.
- 15. ELONGATION OF TRANSLATION Ribosomes elongate the polypeptide chain by a sequential addition of amino acids. The amino acid sequence is determined by the order of the codons in the specific mRNA. Elongation, a cyclic process involving certain elongation factors (EFs), may be divided into three steps:- 1. Binding of aminoacyl t-RNA to A-site. 2. Peptide bond formation. 3. Translocation.
- 16. Binding of aminoacyl—tRNA to A-site: The 80S initiation complex contains met-tRNA1 in the P-site, and the A-site is free. Another aminoacyl-tRNA is placed in the A-site. This requires proper codon recognition on the mRNA and the involvement of elongation factor 1a (EF-la) and supply of energy by GTP. As the aminoacyl-tRNA is placed in the A-site, EF-1a and GDP are recycled to bring another aminoacyl- tRNA. Peptide bond formation: The enzyme peptidyltransferase catalyses the formation of peptide bond (Fig). The activity of this enzyme lies on 28S RNA of 60S ribosomal subunit. It is therefore the rRNA (and not protein) referred to as ribozyme that catalyses the peptide bond formation. As the amino acid in the aminoacyl- tRNA is already activated, no additional energy is required for peptide bond formation.
- 17. Translocation: As the peptide bond formation occurs, the ribosome moves to the next codon of the mRNA (towards 3′-end). This process called translocation, basically involves the movement of growing peptide chain from A-site to P-site. Translocation requires EF-2 and GTP. GTP gets hydrolysed and supplies energy to move mRNA. EF-2 and GTP complex recycles for translocation.
- 18. TERMINATION OF TRANSLATION: • Termination is a simple process when compared to initiation and elongation. After several cycles of elongation, incorporating amino acids and the formation of the specific protein/polypeptide molecule, one of the stop or termination signals (UAA, UAG and UCA) terminates the growing polypeptide.
- 19. INHIBITORS OF PROTEIN SYNTHESIS: Translation is a complex process and it has become a favorite target for inhibition by antibiotics. Antibiotics are the substances produced by bacteria or fungi which inhibit the growth of other organisms. Majority of the antibiotics interfere with the bacterial protein synthesis and are harmless to higher organisms. This is due to the fact that the process of translation sufficiently differs between prokaryotes and eukaryotes. The action of a few important antibiotics on translation is described here.
- 20. Streptomycin: Initiation of protein synthesis is inhibited by streptomycin. It causes misreading of mRNA and interferes with the normal pairing between codons and anticodons. Tetracycline: It inhibits the binding of aminoacyl tRNA to the ribosomal complex. In fact, tetracycline can also block eukaryotic protein synthesis. This, however, does not happen since eukaryotic cell membrane is not permeable to this drug. Puromycin: This has a structural resemblance to aminoacyl tRNA. Puromycin enters the A site and gets incorporated into the growing peptide chain and causes its release. This antibiotic prevents protein synthesis in both prokaryotes and eukaryotes. Chloramphenicol: It acts as a competitive inhibitor of the enzyme peptidyltransferase and thus interferes with elongation of peptide chain.
- 21. Erythromycin: It inhibits translocation by binding with 50S subunit of bacterial ribosome. Diphtheria toxin: It prevents translocation in eukaryotic protein synthesis by inactivating elongation factor eEF2.