Translation - Lecture Notes PDF

BMS: 141 Lecture No: 10 Title: Translation Instructor Name: Dr Lames Dawood Medicine and Surgery Program Fall 2024 1. List Components required for translation. 2. Outline the mechanism of translation. 3. Explai...

BMS: 141 Lecture No: 10 Title: Translation Instructor Name: Dr Lames Dawood Medicine and Surgery Program Fall 2024 1. List Components required for translation. 2. Outline the mechanism of translation. 3. Explain initiation steps of protein synthesis in prokaryotes. 4. Explain elongation steps of protein synthesis in prokaryotes. 5. Explain Termination steps of protein synthesis in prokaryotes. 6. Identify Posttranslational modifications of polypeptide chains.  Outline Translation Components initiation steps of required for protein synthesis translation mechanism of in prokaryotes translation Translation Translation  Outline Translation elongation steps of protein synthesis in Termination modifications prokaryotes steps of protein synthesis in prokaryotes Post Translation Translation  Translation Components required for protein synthesis: 1- Amino acids: all the 20 amino acids involved in the protein must be present at the time of protein synthesis. 2- Ribosomes: the site of protein synthesis. They are large complexes of protein and rRNA.  Types of Ribosomes A- Free Ribosome: Synthesize proteins that remain within the cell cytosol or destined for the nucleus, mitochondria, and peroxisomes. B-Membrane bounded Ribosomes (to ERP): Synthesize proteins that will be secreted from the cells or integrated into plasma, endoplasmic reticulum, or Golgi membranes, or incorporated into lysosomes. C- Polysomes More than one ribosome (about 20) at a time can translate a message on one mRNA simultaneously. Occurs in cells that are carrying out intensive protein synthesis. 3- tRNA: at least one specific type of tRNA is required to transfer one amino acid. There about 31 tRNA in human for the 20 amino acids, this means some amino acids have more than one specific tRNA. 4- aminoacyl-tRNA synthetase: This is the enzyme that catalyzes the attachment of amino acid with its corresponding tRNA forming aminoacyl tRNA. At least 20 specific enzymes are required for the proper attachment of the 20 amino acids to specific tRNA molecules.  Aminoacyl-tRNA synthetases Reactions The enzyme catalyzes a two-step reaction that results in the covalent attachment of the carboxyl group of an amino acid to the 3′-end of its corresponding tRNA. The overall reaction requires (ATP), which is cleaved to (AMP) and inorganic pyrophosphate (PPi).  Amino acid Uncharged tRNA R O = - H2N-C-C-OH 3’ - H ATP Adenylated (activated) amino acid R O = - H2N-C-C-O- AMP PPi - H P P AMP R O Aminoacyl-tRNA = - H2N-C-C-O - synthetases H Reactions Aminoacyl (charged) tRNA Fidelity of Synthetase Enzyme Is due to The extreme specificity of the synthetase in recognizing both the amino acid and its specific tRNA. It has a “proofreading” or “editing” activity that can remove mischarged amino acids from the enzyme or the tRNA molecule.  5-Protein factors: Initiation, elongation, and termination (or release) factors are required for peptide synthesis. 6- ATP and GTP: required as sources of energy. 7- mRNA  TRANSLATION LOADED tRNA COMPONENTS PRESENT IN THE Aminoacid PROCESS carried anticodon codon RIBOSOME mRNA Which of the following identify and move amino acids to the site of protein production? a. rRNA b. tRNA c. iRNA d. mRNA e. snRNA Information in mRNA is translated into a primary sequence of protein in 4 steps: 1 ACTIVATION 2 INITIATION 3 ELONGATION 4 TERMINATION  One important difference between eukaryotics & prokaryotics is that translation and transcription are coupled in prokaryotes, with translation starting before transcription is ended. I- Initiation mRNA carrying the code binds first to the ribosome. mRNA cannot bind directly to the 70 S ribosome in Prok. (80 S in Euk.) ; initiation starts first by binding to smaller subunit (30 S in Prok. , 40 s in Euk.) This is followed by association of the larger subunits (50 S in Prok. or 60 S in Euk.) to form completed initiation complex. Initiation The initiation complex includes: Two ribosomal subunits mRNA to be translated aminoacyl-tRNA specified by the first codon in the GTP message GTP INITIATION FACTORS Initiation factors  Initiation factors & Energy requirements ❑ In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3). ❑ In eukaryotes, there are over ten (Designated eIF to indicate eukaryotic origin). GTP provides energy for the initiation process. Eukaryotes also require ATP for initiation. Mechanisms by which the ribosome recognizes the nucleotide sequence that initiates translation: Shine-Dalgarno sequence: A purine-rich sequence of nucleotide bases is located six to ten bases upstream of the initiating AUG codon on the mRNA molecule—that is, near its 5′-end. 5` mRNA  Shine-Dalgarno sequence The 16S rRNA component of the 30S ribosomal subunit has a nucleotide sequence near its 3′-end that is complementary to the SD sequence in close proximity to the initiating AUG codon. Eukaryotic messages do not have SD sequences, But have a 5` cap. The cap structure at the 5′-end of the mRNA binds to the 40S ribosomal subunit and elF, and moves down the mRNA until it encounters the initiator AUG. This “scanning” process requires ATP.  Initiation codon & tRNA The initiating AUG is recognized by a special initiator tRNA. Recognition is facilitated by IF (bound to GTP), forming pre-initiation complex.  Initiator tRNA In bacteria and in mitochondria, the initiator tRNA carries an N- formylated methionine. (The formyl group is added to the methionine after that amino acid is attached to the initiator tRNA). In eukaryotes, the initiator tRNA carries a methionine that is not formylated. The initiator tRNA enters the ribosomal P site, the large subunit combines and GTP is hydrolyzed to GDP.  Initiation of protein synthesis begins with binding of (A) 40S ribosomal unit on mRNA (B) 60S ribosomal unit on mRNA (C) Charging of tRNA with specific amino acid (D) Attachment of aminoacyl tRNA on mRNA (E) 40S ribosomal unit on tRNA  -Each amino acid in a protein is specified by: A. several genes. B. a promoter. C. a mRNA molecule. D. a codon. E. an enhancer. 🞂 Lippincott Illustrated Review Integrated system 🞂 Lippincott Illustrated Review 6th edition 🞂 Oxford Hand book of Medical Science 2nd edition 🞂 Clinical Key Student BMS: 141 Lecture No: 11 Title: Translation Part 2 Instructor Name: Dr Lamees Dawood Medicine and Surgery Program Fall 2024 1. List Components required for translation. 2. Outline the mechanism of translation. 3. Explain initiation steps of protein synthesis in prokaryotes. 4. Explain elongation steps of protein synthesis in prokaryotes. 5. Explain Termination steps of protein synthesis in prokaryotes. 6. Identify Posttranslational modifications of polypeptide chains. Elongation Elongation is a cyclic process on the ribosome. AA is added to the carboxyl end of growing peptide according to the order of the codons in the mRNA. The ribosome moves from the 5′-end to the 3′-end of the mRNA that is being translated. Elongation requires GTP hydrolysis & Elongation factors.  Elongation The Three Steps of Elongation 1] Binding of an Incoming Aminoacyl-tRNA 2] Peptide Bond Formation 3] Translocation  Elongation 1. Binding of an Incoming Aminoacyl-tRNA Elongation factors (EF) help binding of the proper aminoacyl-tRNA complementary to the codon on A site. It is energy consuming reaction utilizing GTP.  2- Peptidyl transferase A component of 50S transfers the AA from the P site onto the AA at the A site, and catalyzes peptide bond formation.  P A peptide bond formation Result in the formation of a polypeptide linked to tRNA at “A” site leaving uncharged tRNA in “P” site. The energy for peptide bond formation comes P A from the ATP used in tRNA charging.  3- Translocation After the peptide bond has been formed, the ribosome advances three nucleotides toward the 3′-end of the mRNA. This is catalyzed by elongation factors EF, in prokaryotes and use eEF in eukaryotic cells. GTP hydrolysis is needed in this step.  Translocation results in 1- Movement of uncharged tRNA to “E” site and subsequent separation. 2- Movement of the peptidyl tRNA to occupy by “P” site. 3- Now the “A” site is empty and expose the next codon that attract next amino acyl tRNA. Translation - animation Extended Modular Program Elongation (Adding New Amino Acids) Codon recognition Peptide bond formation Translocation: ribosome moves along mRNA, aminoacyl tRNA shifts from A site to P site  Termination Termination occurs when one of the three termination codons moves into the A site. These codons are recognized by release factors:  Termination The binding of release Factors induces Peptidyl transferase to hydrolyze the bond linking the peptide to the tRNA, causing the nascent protein to be released from the ribosome. GTP is hydrolyzed in the this termination step, the release factors are free, mRNA is released as well as well as the two ribosomal subunits. Termination A stop codon is reached UAA UAG UGA All parts release  ANIMATION Watch this simplified animation: https://www.youtube.com/watch?v=gG7uCskUOrA&t=16 s When does protein synthesis stop? A. When there is enough protein. B. When the stop codon enters the ribosome. C. When the stop anticodon is located on the mRNA. D. When the intron has been replaced. When does protein synthesis stop? A. When there is enough protein. B. When the stop codon enters the ribosome. C. When the stop anticodon is located on the mRNA. D. When the intron has been replaced. Regulation of translation Regulation in eukaryotes is by covalent modification of eIF-2 (phosphorylated eIF-2 is inactive). Certain proteins bind mRNA and either inhibit its use by blocking translation or extend its use by protecting it from degradation. POSTTRANSLATION MODIFICATION 1. Protein targeting Many protein synthesis occurs on polysomes in the cytosol of eukaryotic cells, and remain in the cytosol, where they carry their function.  Protein targeting The newly synthesized polypeptides pass to destined compartment due to presence of a signal sequence or targeting sequences (sometimes called a leader sequence or localization signal) that direct these proteins to their final locations.  2. Protein Folding Mentioned in the chapter of amino acids and proteins  Post-translational Modifications Of proteins Occur, while proteins are still attached to the ribosome or after their synthesis has been completed. Failure of post-translation modification; affect the normal function of many proteins.  3. Trimming Many proteins are initially made as large, precursor molecules that are not functionally active. Portions of the protein chain must be removed by specialized endoproteases, resulting in the release of an active molecule. Trimming or Proteolytic cleavage 1- The initiating N- terminal methionine is cleaved from the primary translation product. 2- Signal sequences are removed by specific peptidases. 3- Proteolytic removal of polypeptides for activation of proteins Hormones; e.g. pro-insulin, pro Collagen Enzymes; e.g.(zymogen) chymotrypsinogen and trypsinogen. 4. Covalent Modification Proteins, both enzymatic and structural, may be modified by the covalent attachment of a variety of chemical groups. a. Phosphorylation Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less frequently, tyrosine residues in a protein. Catalyzed by kinases and may be reversed by phosphatases.  b. Glycosylation Carbohydrate chains attached to serine or threonine hydroxyl groups (O-linked) or the amide nitrogen of asparagine (N- linked). Occurs in the endoplasmic reticulum and the Golgi apparatus. Sometimes it is used to target proteins to specific organelles as lysosome  C. Hydroxylation Proline and lysine residues of the α chains of collagen are extensively hydroxylated in the endoplasmic reticulum.  5. Protein degradation Proteins that are defective or destined for rapid turnover are often marked for destruction by ubiquitination (attachment of ubiquitin). Ub is a small protein (76 AA). Proteins marked in this way are rapidly degraded by a cellular component known as the “proteasome,” using ATP.  SUMMARY -Components required for protein synthesis are: Amino acids, Ribosomes, tRNA , aminoacyl-tRNA synthetase, Protein factors and energy. -steps of protein synthesis are initiation, elongation and Translation. -Initiation of Translation includes assembly of the components of the translation system before peptide bond formation occurs.  Summary - steps of protein synthesis are initiation, elongation and Translation. - Ribosomes have 3 sites: Peptidyl-tRNA binding site (P), Aminoacyl- tRNA binding site (A) and exit site (E). - Posttranslational modification of polypeptide chains is covalent modification or trimming. - Proteins that are defective or destined for rapid turnover are often marked for destruction by ubiquitination. Extended Modular Program 🞂 Lippincott Illustrated Review Integrated system 🞂 Lippincott Illustrated Review 6th edition 🞂 Oxford Hand book of Medical Science 2nd edition 🞂 Clinical Key Student