FTM 14 - Translation and Post Translational Modification.pdf

Basic Principles of Medicine 1 Module: Foundations to Medicine Lecture No: 14 Lecture Title: Translation and Post-translational Modification Dr Bert van Loo [email protected] or [email protected] Slides created by Dr Cristofre Martin from St Georges University © Copyright All year 1 courses...

Basic Principles of Medicine 1 Module: Foundations to Medicine Lecture No: 14 Lecture Title: Translation and Post-translational Modification Dr Bert van Loo [email protected] or [email protected] Slides created by Dr Cristofre Martin from St Georges University © Copyright All year 1 courses materials, whether in print or online, are protected by copyright. The work, or parts of it, may not be copied, distributed or published in any form, printed, electronic or otherwise. As an exception, students enrolled in year 1 of St. George’s University School of Medicine and their faculty are permitted to make electronic or print copies of all downloadable files for personal and classroom use only, provided that no alterations to the documents are made and that the copyright statement is maintained in all copies. View only files, such as lecture recordings, are explicitly excluded from download and creating copies of these recordings by students and other users are strictly illegal. The author of this document has made the best effort to observe current copyright law and the copyright policy of St George's University. Users of this document identifying potential violations of these regulations are asked to bring their concern to the attention of the author. Translation and post-translational modifications Lecture Objectives SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Describe the sequence of events that occurs during translation in prokaryotes (initiation, elongation and termination) and list the major differences between prokaryotic and eukaryotic translation. SOM.MK.I.BPM1.1.FTM.3.BCHM.0074 Explain how diphtheria toxin interferes with eukaryotic translation. Explain the role of miRNA as inhibitors of translation. SOM.MK.I.BPM1.1.FTM.3.BCHM.0069 Explain the mode of action of common antibiotics that interfere with translation: Initiation inhibitors (streptomycin), Elongation inhibitors (Tetracycline, chloramphenicol, erythromycin, puromycin, cycloheximide). SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 Review examples of post-translational modifications: Zymogen activation (trypsinogen to trypsin), Serine/threonine phosphorylation (regulation of enzymes in metabolism), Tyrosine phosphorylation (Insulin receptor), O-linked glycosylation, N-linked glycosylation and Lipid anchoring (farnesyl groups to Ras). SOM.MK.I.BPM1.1.FTM.3.BCHM.0076 Describe proteolytic processing and post-translational modifications using insulin as an example. Pre-lecture Reading View Panopto videos: Genetic Code and Translation Recommended Reading • Lippincott’s Biochemistry: Chapter 32 Protein Synthesis | Lippincott® Illustrated Reviews: Biochemistry, 8e | Medical Education | Health Library (lwwhealthlibrary.com) • Questions: 32.1-32.3, 32.5-32.9 SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Additional Resources YouTube videos: • The big picture of translation: • http://www.youtube.com/watch?v=5bLEDd-PSTQ • 3 minutes 32 seconds. • Interpreting the genetic code: • https://www.youtube.com/watch?v=-Ht81lHiJac • 4 minutes 16 seconds. SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Translation Formation of a polypeptide polymer from an RNA template ➢ mRNA ➢ Translated to ➢ Polypeptide SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Translation: Requirements • The biological polymerization of amino acids into a polypeptide chain (Translation) requires: • Messenger RNA (mRNA) • Ribosomes • Charged transfer RNA (tRNA) • Initiation factors • GTP SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Schematic Model of Ribosome with mRNA, tRNA and growing Polypeptide chain Translation: The Process 1. 2. 3. 4. 5. Activation of the monomer Initiation Elongation Termination Processing the polymer Translation: the RNA-directed synthesis of a polypeptide We will describe translation in prokaryotes and provide notes regarding the eukaryotic system when appropriate. SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 9 Amino acids are activated by attachment to tRNA • Charging the tRNA is a two step process: 1. Enzyme bound amino-acid-adenylate 2. Formation of the aminoacyl-tRNA • Reaction is driven by hydrolysis of pyrophosphate • tRNA to which amino acid is attached is called the “charged tRNA” (Charged tRNA) Copyright © Wolters Kluwer. SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 10 Generation of the initiator N-formylmethionyl-transfer RNA (fMet-tRNAi). • N-formylmethionine (fMet) as the first amino acid – in prokaryotes & in mitochondria. • This special tRNAi (fMet-tRNAi) is recognized differently by the ribosome – allows initiation • Prokaryotes have 2 tRNAs for methionine: • one allows formation of fMet, • the other recognizes internal AUG codons. Copyright © Wolters Kluwer. SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Use of Special Met-tRNAi as Initiator tRNA The idea is to get the first MET containing tRNAi into the P-site to allow initiation • Prokaryotes & Mitochondria of Eukaryotes: • Two tRNAs that recognize AUG 1. Formylated MET for first codon 2. Normal Met-tRNA for internal codons • Eukaryotes: • Two tRNAs that recognize AUG 1. The first codon also uses MET, and it has a special tRNA for this first codon (but the MET amino acid is not formylated). 2. Normal Met-tRNA for internal codons special recognize Initiator tRNA always carries methionine 12 SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Correct Alignment of the AUG codon with respect to the ribosome Complementary binding between prokaryotic mRNA Shine–Dalgarno sequence and 16S rRNA. Copyright © Wolters Kluwer. • Prokaryotes: Shine Dalgarno sequence is purine rich and resides a few (5-10) bases 5’ to the start codon • Eukaryotes: lack Shine Dalgarno sequence, therefore eukaryotic small ribosome binds close to the cap at the 5’ end, scans until it encounters the AUG start codon. SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Protein Synthesis 1 Initiation factors (IFs) aid in the formation of the 30S initiation complex. The charged initiator tRNA is brought to the P site of the 30S subunit by IF-2-GTP. INITIATION 2 GTP on IF-2 is hydrolyzed and initiation factors are released when the 50S subunit arrives to form the 70S initiation complex. SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Protein Synthesis ELONGATION 3 Elongation factor EF-Tu-GTP brings the appropriately charged tRNA to the codon in the empty A site (decoding). GTP on EF-Tu is hydrolyzed. 4 Peptidyltransferase activity of the 23S rRNA of 50S subunit catalyzes peptide bond formation, transferring the initiating amino acid (or peptide chain) from the P site to the amino acid at the A site (transpeptidation). Enzymatic activity of the RNA portion of the 50S ribosome – a Ribozyme SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Protein Synthesis 5 EF-G-GTP facilitates movement of the ribosome three nucleotides along the mRNA in the 5’ to 3’ direction. What was in P site is now in E site, what was in A site is now in P site, and A site is empty. GTP on EF-G is hydrolyzed. TRANSLOCATION Translocation • EF-G is the prokaryotic protein • In eukaryotes, EF-G is called EF-2 SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 6 Steps 3, 4, and 5 are repeated until a termination codon is encountered at the A site. Protein Synthesis TERMINATION • Stop codon has no tRNA • Stop codon causes elongation to stall • Peptide is held in the P-site attached to the tRNA • Release Factor (RF) diffuses into the A-site • RF allows peptidyl transferase to cleave ester bond between tRNA and the peptide • After release of the peptide, the ribosome dissociates into its subunits which may then begin a new round of translation. SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 7 A termination codon is recognized by a release factor (RF-1 or RF-2), which results in release of the newly synthesized protein. GTP on RF-3 is hydrolyzed. The synthesizing complex dissociates. A Polyribosome: ribosomes simultaneously translating one mRNA • A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome) • Polyribosomes enable a cell to make many copies of a polypeptide very quickly. SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Mobile Session ID: docmartin Transcription and Translation is Coupled in Prokaryotes DNA Electron microscopy showing coupled transcription and translation in prokaryotes Short transcripts as RNA Polyribosome formation longer transcripts as RNA polymerase begins on the RNA transcript polymerase nears the end transcription of the gene Proteins are not labeled, so we don’t see them SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 25 Protein Folding • Protein folding is the process whereby proteins acquire their mature functional (native) structure. • Often begins cotranslationally • Occurs spontaneously or facilitated by chaperones. Chaperones ensure that only a limited number of folds are available to a newly synthesized protein. 26 SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Some major differences between Prokaryotic & Eukaryotic Translation Feature Prokaryotes Eukaryotes Initiator tRNA Formylated Special, but not formylated RNA Polycistronic Monocistronic Translation start site May select an internal AUG Typically start translation at first AUG site Transcription and Translation Coupled Not coupled because of nuclear membrane Typically, methionine is NOT found as the first amino acid on a mature protein – post translationally modified 27 SOM.MK.I.BPM1.1.FTM.3.BCHM.0073 Compounds Affecting Protein Synthesis • Diphtheria toxin inactivation of EF-2 by ADP-ribosylation Characteristic membranous pharyngitis Toxin A is produced by a lysogenic bacteriophage that infects Corynebacterium diphtheriae. The toxin Catalyzes the transfer of ADP-ribose to host cells EF-2, inactivating it (prevents translocation) & inhibiting protein synthesis. • Antibiotic Inhibition of initiation • Streptomycin (aminoglycoside) • Prevents assembly of ribosome (binds to 30s subunit) • Antibiotic Inhibition of elongation • Tetracycline - four (tetra) ring (cyclic) structure • Block elongation by preventing aminoacyl-tRNA access to the A-site • Erythromycin (macrolide) • Binds to the 50S subunit of the complete (70S) ribosome • Blocks ribosome translocation • Chloramphenicol • Inhibits peptidyl transferase activity in prokaryotes • At high levels, may inhibit mitochondrial translation • Cycloheximide • Inhibits eukaryotic peptidyl transferase activity • Puromycin • Causes premature termination of translation in both prokaryotes and eukaryotes SOM.MK.I.BPM1.1.FTM.3.BCHM.0074 There are many more 28 SOM.MK.I.BPM1.1.FTM.3.BCHM.0069 Inhibitors of Protein Synthesis SOM.MK.I.BPM1.1.FTM.3.BCHM.0074 SOM.MK.I.BPM1.1.FTM.3.BCHM.0069 Inhibitors of Protein Synthesis -continued • EF-G is the prokaryotic protein • In eukaryotes, EF-G is called EF-2 SOM.MK.I.BPM1.1.FTM.3.BCHM.0074 SOM.MK.I.BPM1.1.FTM.3.BCHM.0069 RNA Interference (RNAi) & Inhibition of Translation • miRNA as Inhibitors of Translation: The extent of complementarity between the target mRNA and the miRNA determines the final outcome: • Perfect complementarity results in mRNA degradation. • Important role in processes like cell proliferation, differentiation, and programmed cell death (apoptosis). • Treatment of hereditary transthyretin-mediated amyloidosis (hATTR) by RNAi: In 2018, the first RNAi-based therapy was approved to treat peripheral nerve disease (polyneuropathy) in patients with hATTR due to a mutation in the gene encoding transthyretin (TTR). • The siRNA-based drug, patisiran, prevents the production of abnormal TTR protein and reduces the buildup of amyloid deposits containing TTR that form in peripheral nerves and in the heart. SOM.MK.I.BPM1.1.FTM.3.BCHM.0074 SOM.MK.I.BPM1.1.FTM.3.BCHM.0050 Production and action of microRNAs (miRNA) Pri = primary; RISC = RNA-induced silencing complex. Lippincott Biochemistry p525 Post-translational Modifications of Proteins • Some proteins are released from the ribosome nearly ready to function, while others undergo a variety of post-translational modifications. • Post-translational modification may occur as the polypeptide is being translated or after translation is completed and may be reversible or irreversible. • Post-translational modification of a protein may result in its conversion to a functional form, its direction to a specific sub-cellular compartment, its secretion from the cell, alteration of its activity or stability. • Major types of post-translational modifications: 1) Protein folding (e.g. formation of disulphide bridges). 2) Covalent alterations (e.g. phosphorylation, glycosylation, hydroxylation). 3) Proteolytic processing (trimming: e.g. zymogen activation). 4) Addition of prosthetic groups (e.g. Heme of hemoglobin). 5) Prenylation (e.g. lipid anchoring, farnesylation). 6) Protein degradation (e.g. ubiquitination and degradation by the proteasome complex) • We will discuss a few! 36 SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 SOM.MK.I.BPM1.1.FTM.3.BCHM.0076 Zymogen Activation • Zymogen (or proenzyme): an inactive enzyme precursor • Activated within an organism into active enzymes • Activation by enzymatic cleavage of peptide bonds of the zymogen molecule • Cascade of zymogen activation: • Caspases to activate apoptosis • blood coagulation • Digestion of proteins Cleavage of dietary protein in the small intestine by pancreatic proteases (zymogens). SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 Protein Phosphorylation by a Kinase ✓ Phosphate is transferred from ATP to an amino acid side chain Tyrosine ✓ Occurs on the OH groups of serine, threonine, or less frequently tyrosine residues. ✓ Phosphorylation is the most common posttranslational modification in eukaryotes: may be permanent or reversible. Dephosphorylation by phosphatases ✓The Insulin Receptor (IRs) is a tyrosine kinase 38 SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 Glycosylation: alters the properties of proteins, changing their stability, solubility & physical bulk. • O – linked • Carbohydrate chain attached to the OH group of Ser/Thr • Glycan groups always face extracellular side • Occurs only after the protein reaches the Golgi Apparatus • N – Linked • Carbohydrate chains attached to the amide nitrogen of Asn residue. • Occurs in the ER & Golgi. In ER, modulates folding of proteins. • The carbohydrate moieties act as recognition signals: • Protein is targeted to either the plasma membrane, to organelle interiors or organelle membranes. • Influence cell-cell interactions. • Involved in the development of an organism. Catalyzed by Glycosyltransferases 39 SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 Lipid Anchoring Extracellular • The cell targets Ras protein to the cytosolic face (inner leaflet) of the plasma membrane by a lipid anchor mechanism - with the aid of farnesyl groups. Farnesyl group S Protein Ras Cytosol SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 • Farnesyl is a 15-carbon isoprenoid group which may be attached to cysteine. 40 Proteolytic Processing of Insulin • Insulin is derived from a single polypeptide • A chain = 21 amino acids, • B chain = 30 amino acids • Insulin binds to Insulin Receptor (tyrosine kinase) to signal that blood glucose is high • C-peptide is essential for proper insulin folding • C-peptide is a good indicator of insulin production and secretion because its (C-peptide) half-life in the plasma is longer than that of insulin. • Measurement of the C-peptide levels has a clinical value (discussed later in the course) SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 SOM.MK.I.BPM1.1.FTM.3.BCHM.0076 Proteins destined for secretion are translated into the rER Signal sequence SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 SOM.MK.I.BPM1.1.FTM.3.BCHM.0076 3. Translation of the polypeptide is directed into the lumen of the rER and forms “preproinsulin” 4. The signal sequence is cleaved in the lumen of the rER – forms “proinsulin” Maturation of insulin occurs in the golgi • Insulin is secreted by the β-cell in response to high blood glucose • Insulin and its C-peptide are packaged together into secretory vesicles to be made ready for secretion • Insulin and the C-peptide are released into the blood • There might be (?) a cellular role for the Cpeptide SOM.MK.I.BPM1.1.FTM.3.BCHM.0075 SOM.MK.I.BPM1.1.FTM.3.BCHM.0076 Thank you! Dr Bert van Loo: [email protected] or [email protected]