Lecture 4 Translation PDF

Translation (Protein Synthesis) Prof. Dr. Ghada Ahmed Abdel-Aleem 1  Objectives: Recognition of the genetic code, how they specify the order of amino acids during protein synthesis. Identification of genetic code characters. Comparing between different types of DNA mutation and the...

Translation (Protein Synthesis) Prof. Dr. Ghada Ahmed Abdel-Aleem 1  Objectives: Recognition of the genetic code, how they specify the order of amino acids during protein synthesis. Identification of genetic code characters. Comparing between different types of DNA mutation and the resulting genetic diseases. Identification of Requirements of protein synthesis including ; Ribosomes, mRNA, Amino acids, tRNA, Aminoacyl-tRNA synthetase enzyme Outline the Steps of protein synthesis: “Translation” Activation, Initiation, Elongation, Termination Studying examples of Inhibitors of protein synthesis Recognition of Post-translational processing of proteins 2  Overview Genes in DNA contain information to make proteins. The cell makes mRNA copies of genes that are needed. The mRNA is read at the ribosomes in the rough ER. Protein is produced. 3  Translation Definition: The synthesis of protein using mRNA as the template, in other words, to translate the nucleotide sequence of mRNA into the amino acid sequence of protein according to the genetic code. 4  GENETIC CODE The genetic code is a “dictionary” that specifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual “word” in the code is composed of three nucleotide bases. These genetic words are called codons. CODON Codon is the sequence of 3 nucleotide bases on mRNA that determines the type and position of amino acid on a protein. There are 4 different nucleotides in the mRNA. As the genetic codon is triplet, there are (4)3 = 64 codons. 5 6 Sixty-one codons code for the 20 amino acids in nature. For example, the codon AUG codes for methionine [Met]. (Note: AUG is the initiation [starting] codon for translation) Three Nonsense (termination) codons: (UAA, UAG and UGA) don't code for amino acids and are called nonsense codons or termination codons. When one of these codons appears in an mRNA sequence, they terminate the translation process. 7 TheGeneticCodeis degenerate( >1 codon/aminoacid ) 8 9 10 11 12  CLINICAL SIGNIFICANCE Mutations are permanent changes in DNA sequence. Mutations lead to new hereditary variations and new gene is called a mutant. Insertion: One or more bases is inserted into the DNA strand. 13 Deletion: One or more bases is deleted from the DNA strand. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 1- Which one of the following has the anticodon? A. ribosomal RNA B. tRNA C. RNA polymerase D. mRNA 2- Which one of the following is CORRECT about the Genetic code? A) Overlapping B) Non-overlapping C) Not universal D) Ambiguous 30 1- Which one of the following has the anticodon? A. ribosomal RNA B. tRNA C. RNA polymerase D. mRNA 2- Which one of the following is CORRECT about the Genetic code? A) Overlapping B) Non-overlapping C) Not universal D) Ambiguous 31 32  TRANSLATION ⚫ Production of proteins from mRNA(read in the 5  to 3  direction by ribosomes) ⚫ mRNA goes to the ribosomes in the cytoplasm or the RER and produces proteins 33  Requirements of protein synthesis 34 The synthesis of a certain protein requires: A ribosome, the protein synthesizing machinery. mRNA, which carries the information needed for arranging the amino acids in the proper order of the specific protein. Amino acids, the building units of the protein.(20) tRNA, which carries the amino acids to the proper place in the polypeptide chain. Aminoacyl-tRNA synthetase enzyme, which connects the amino acids to the specific carrier tRNA. A source of energy, in the form of ATP for connecting amino acids to the tRNA, and GTP for the interaction of aminoacyl- tRNAs with the ribosomes. Mg+2 Protein factors, which are required for the interaction of the ribosome with mRNA and tRNA. (IF, EF, TF) 35 THE RIBOSOME Ribosomes are large complexes of protein and rRNA. They consist of two subunits one large and one small whose relative sizes are generally given in terms of their sedimentation coefficients, or S (Svedberg) values. The prokaryotic 50S and 30S ribosomal subunits together form a ribosome with an S value of 70. The eukaryotic 60S and 40S subunits form an 80S ribosome. prokarytic ribosome Eukaryotic ribosome 70S 80S 5050S S 60S 30S 30 S 40S 5.8S 5S RNA RNA 5S RNA 23S 16S 28S 18S RNA RNA RNA RNA 36 32 Proteins 21 Proteins ~50 Proteins ~30 Proteins  P site A site E site 37  MESSENGER RNA The mRNA is composed of 3 successive sequences, a 5’ nontranslated leading sequence, a translated coding sequence, and a 3’ nontranslated trailing sequence that usually ends in a poly (A) tail. Initiating Terminating codon codon AUG UAA 5’ 3’ Nontranslated Translated coding sequence Nontranslated leading sequence trailing sequence 38  5’-End 3’-End 3 2 1 5’-End 1 2 3 3’-End 39 Activation of tRNA (AMINOACYL-tRNA SYNTHETASE) In the cytosol, there are 20 species of this enzyme, one specific for each of the 20 amino acids required for protein synthesis. This enzyme connects the carboxyl group of the amino acid to the 3’OH of the specific tRNA. The enzyme recognizes the R radical of the amino acid and recognizes the anticodon of the tRNA. Activation of the amino acid occurs in 2 steps: – 1) The amino acid reacts with ATP, forming enzyme-bound aminoacyl-AMP. A pyrophosphate is liberated. – 2) The enzyme transfers the amino acid to the specific tRNA, forming aminoacyl-tRNA. AMP and the free enzyme are liberated. 40  Amino acid Aminoacyl-tRNA ATP synthetase PPi AMPAmino acid CCA AMP CCAAmino acid 41 Aminoacyl-tRNA  STEPS IN PROTEIN SYNTHESIS: “TRANSLATION” 42  Stagesof Proteinbiosynthesis ① Activation of Amino Acids ② Initiation: ③ Elongation: ④ Termination ⑤ Folding and Posttranslational Processing 43  A. Initiation It requires that an mRNA molecule be selected for translation by ribosome and binds it. Once binding occur, the ribosome finds the reading frame and begins the translation. Requirements of initiation: [tRNA, Ribosome, mRNA, amino acids, GTP and ATP, and at least 10 eukaryotic initiation factors (eIFs) that facilitate the assembly of the initiation complex]. 44 The small ribosomal subunit binds to the mRNA. In prokaryotes, the 16S rRNA of the small subunit binds to the Shine-Dalgarno sequence in the 5′- untranslated region of the mRNA. In eukaryotes, the small subunit binds to the 5′ cap structure and slides down the message to the first AUG. Messenger RNA Shine-Dalgarno 3’ sequence 3’ 5’ 5’ 16S-Ribosomal RNA 30S-Ribosomal subunit 45  2- Initiation codon – The charged initiator tRNA becomes bound to the AUG start codon on the message through base pairing with its anticodon. – The initiator tRNA in prokaryotes carries N- formylated methionine (fMet), whereas the initiator tRNA in eukaryotes carries Methionine (not formylated). – The large subunit binds to the small subunit, forming the completed initiation complex. 46  P E A fMET UAC mRNA GDP + Pi 47  B. Elongation A. Binding of Aminoacyl-tRNA at the A Site – Binding of the codon and the anticodon occurs by base-pairing and is antiparallel. – Binding of the aminoacyl-tRNA to the A site requires the action of an elongation factor and the hydrolysis of GTP to GDP and Pi. B. Formation of a Peptide Bond – The amino acid at the A site forms a peptide bond with the amino acid attached to the tRNA at the P site. – Formation of the peptide bond is catalyzed within the peptidyl transferase site by the help of 23S rRNA in the large ribosomal subunit. – The tRNA at the P site now does not contain an amino acid. It is “uncharged.” – The growing polypeptide chain is attached to the tRNA in the A site. 48  C. Translocation by TRANSLOCASE enzyme – After the peptide bond has been formed, the ribosome advances three nucleotides toward the 3’-end of the mRNA. This process is known as translocation and, requires the participation of EF-G and the hydrolysis of GTP (elongation factor 2, EF-2,in eukaryotes). – This causes movement of the uncharged tRNA into the ribosomal E site (before being released) and movement of the peptidyl-tRNA into the P site. – The elongation and translocation steps are repeated 49 until a termination codon moves into the A site.  Phe P E A fMET AAA UAC mRNA EF-Tu GDP + Pi EF-Ts 50  Peptide bond fMET Phe UAC AAA 51  fMET Phe UAC AAA GDP + Pi EF-G 52  fMET Phe Lys AAA U U C EF-Tu GDP + Pi EF-Ts 53  fMET Phe Lys AAA U U C GDP + Pi EF-G 54 C. Termination Termination occurs when one of the three termination codons moves into the A site. These codons are recognized by TERMINATION factors. (Releasing factors). RF-1 recognizes the termination codons UAA and UAG and UGA. The complex formed of RF-1, RF-3 and GTP with peptidyl transferase enzyme promotes the hydrolysis of the bond between peptide and tRNA occupying the P site 55  Arg GDP + Pi UU C Termination codon 56 1.Which one of the followings is an example of frame shift mutation? A- Sickle cell anemia B- Cystic fibrosis C-Xeroderma pigmentosum. D- Colorectal cancer. 2- The enzyme amino acyl tRNA synthetase is involved in 3- Which of the following molecules is A. Dissociation of discharged tRNA NOT a component of the 30S initiation from 80S ribosome complex? B. Charging of tRNA with specific (A) GTP amino acids (B) mRNA C. Termination of protein synthesis (C) ATP (D) initiating factor 2 D. Nucleophilic attack on esterified carboxyl 57 group of peptidyl tRNA 1.Which one of the followings is an example of frame shift mutation? A- Sickle cell anemia B- Cystic fibrosis C-Xeroderma pigmentosum. D- Colorectal cancer. 2- The enzyme amino acyl tRNA synthetase is involved in 3- Which of the following molecules is A. Dissociation of discharged tRNA NOT a component of the 30S initiation from 80S ribosome complex? B. Charging of tRNA with specific (A) GTP amino acids (B) mRNA C. Termination of protein synthesis (C) ATP (D) initiating factor 2 D. Nucleophilic attack on esterified carboxyl 58 group of peptidyl tRNA 1.Which one of the followings is an example of frame shift mutation? A- Sickle cell anemia B- Cystic fibrosis C-Xeroderma pigmentosum. D- Colorectal cancer. 2- The enzyme amino acyl tRNA synthetase is involved in 3- Which of the following molecules is A. Dissociation of discharged tRNA NOT a component of the 30S initiation from 80S ribosome complex? B. Charging of tRNA with specific (A) GTP amino acids (B) mRNA C. Termination of protein synthesis (C) ATP (D) initiating factor 2 D. Nucleophilic attack on esterified carboxyl 59 group of peptidyl tRNA  INHIBITORS OF TRANSLATION 1. Streptomycin – Streptomycin is an antibiotic that binds with the 30Sribosomal subunit of bacteria, causes misreading of mRNA, and thereby prevents formation of the initiation complex. 2. Tetracycline – Tetracycline is an antibiotic that binds with the 30S ribosomal subunit of bacteria, Tetracycline binds to the 305 ribosomal subunit of prokaryotes and inhibits binding of aminoacyl-tRNA to the A site. 3. Chloramphenicol – Chloramphenicol is an antibiotic that inhibits prokaryotic peptidyltransferase activity of the 50S ribosomal subunit. High doses inhibit mitochondrial ribosomal enzyme. 60 4. Erythromycin and clindamycin This antibiotic binds to the 50S ribosomal subunit of bacteria, inhibiting translocation of peptidyl-tRNA. 5. Puromycin Puromycin is structurally similar to tyrosyl-tRNA. It binds at the A site, forms a peptide bond with the growing peptide chain, causing premature termination of its synthesis both in prokaryotes and in eukaryotes. It cannot be used as antibiotic for treating infections. 6- Ricin (from castor beans) is a very potent toxin that exerts its effects by removing an adenine from 28S ribosomal RNA, thus inhibiting eukaryotic ribosomes. 7- Diphtheria toxin inactivates eukaryotic elongation factor.e EF-2 thus preventing translocation. 61  POST-TRANSLATIONAL PROCESSING OF PROTEINS 62  I. TRIMMING Trimming means removal of part of the peptide chain. II. COVALENT MODIFICATION 1. Phosphorylation – This is very important in the activation-inactivation of many enzymes. 2. Glycosylation – Targets protein to become a part of plasma membrane, lysosomes, or to 63 be secreted out of cells  II. COVALENT MODIFICATION (Cont.) 3. Acetylation – Acetyl radicals may be connected to the є- amino group of lysine. This is very important in histones as it leads to its separation from DNA, which becomes transcriptionally active. 4. Hydroxylation – Prolyl and lysyl residues are hydroxylated in collagen. This is very important for hydrogen bonding and formation of strong collagen. 5. Carboxylation – Glutamate residues may be carboxylated in some proteins, e.g., osteocalcin, protein C, protein S, prothrombin, clotting factors VII, IX, and X. This helps these proteins to bind calcium to perform their function. III. Folding into tertiary and quaternary conformations to be active. 64  1-Enumerate four (4) types of covalent modifications that may occur to the newly synthesized polypeptide in the process of in post- translational modifications. 2- In prokaryotes,Tetracylin prevents synthesis of polypeptide chain by A. Blocking mRNA formation from DNA B. Releasing peptides from mRNA-tRNA complex C. Competing with mRNA for ribosomal binding sites D. Preventing binding of aminoacyl tRNA to A site 65  1-Enumerate four (4) types of covalent modifications that may occur to the newly synthesized polypeptide in the process of in post- translational modifications. 2- In prokaryotes,Tetracylin prevents synthesis of polypeptide chain by A. Blocking mRNA formation from DNA B. Releasing peptides from mRNA-tRNA complex C. Competing with mRNA for ribosomal binding sites D. Preventing binding of aminoacyl tRNA to A site 66  Reference Harvey, Richard A., Ph. D. (2017). Lippincott's illustrated reviews: Biochemistry. Philadelphia :Wolters Kluwer Health, 7th edition 67 68