Natural Sciences > Biology > Antibiotics, Toxins, and Protein Engineering

Antibiotics, Toxins, and Protein Engineering posted by duggu on 12/8/2007

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Course Description

The lethal poison Ricin (best known as a weapon of bioterrorism), Diphtheria toxin (the causative agent of a highly contagious bacterial disease), and the widely used antibiotic tetracycline have one thing in common: They specifically target the cell's translational apparatus and disrupt protein synthesis.

In this course, we will explore the mechanisms of action of toxins and antibiotics, their roles in everyday medicine, and the emergence and spread of drug resistance. We will also discuss the identification of new drug targets and how we can manipulate the protein synthesis machinery to provide powerful tools for protein engineering and potential new treatments for patients with devastating diseases, such as cystic fibrosis and muscular dystrophy.

This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

Syllabus

Prerequisites

A basic knowledge of the central dogma of molecular biology: DNA → RNA → protein.

Overview

The lethal poison Ricin - best known as a weapon of bioterrorism, Diphtheria toxin - the causative agent of a highly contagious bacterial disease, and the widely used antibiotic tetracycline have one thing in common: They specifically target the cell's translational apparatus and disrupt protein synthesis.

In this course, we will explore the mechanisms of action of toxins and antibiotics, their roles in everyday medicine, and the emergence and spread of drug resistance. We will also discuss the identification of new drug targets, and how we can manipulate the protein synthesis machinery to provide powerful tools for protein engineering and potential new medical treatments for patients with devastating diseases such as cystic fibrosis and muscular dystrophy.

Protein synthesis is a fundamental and ancient process that has been highly conserved in all cells from bacteria to human. Protein synthesis takes place on the ribosome and requires many protein and RNA factors to ensure timely and accurate translation of the mRNA. Because of the essential and ubiquitous role of proteins in all cells, protein synthesis has been a prime target of antibiotics and toxins that can effectively shut down translation and cell function. Bacteria, fungi and plants produce various compounds that are extremely potent inhibitors of translation and therefore toxic to cells. The overall mechanism of protein synthesis is similar in all kingdoms of life. However, there are some essential structural differences between the protein synthesis machineries of bacteria and eukaryotes that allow for highly selective inhibition of protein translation in one kingdom but not another. This selective inhibition has important medical implication, as it is the basis of antibiotic usage to combat bacterial infections.

In this course, we will cover protein translation in detail and discuss the scientific literature that investigates the mechanism of action of protein synthesis inhibitors such as Ricin, Diphtheria toxin, and some major antibiotics (e.g. tetracycline). We will also learn about diseases caused by various defects in the translational machinery and possible new medical treatments. Finally, we will discuss cutting-edge methods that are currently being developed to "twist and tweak" normal protein synthesis for the purpose of protein engineering.

Course Objectives

The main objective of this course is to familiarize students with the primary scientific literature (in print and on-line databases) while you discover the exciting world of protein synthesis. You will learn how to read, analyze and critically evaluate scientific papers, and you will be encouraged to engage in active class discussions. The course will provide fundamental insight into the structure and function of the ribosome, its role in protein synthesis, structure and function of the ribosome, and mechanism of action of antibiotics and toxins that target translational machinery. A strong focus will be on the methodology and experimental approaches used, from basic biochemistry, genetics, and molecular biology, to state-of-the-art protein engineering.

Format and Expectations

Most meetings will consist of discussion of two research papers. Students are expected to have read the papers in advance, use on-line literature databases for optional background reading, and be prepared to discuss the selected papers in class. At the end of each class, the necessary background to understand the papers for the next session will be provided.

Assignments

There are two projects on the following topics:

Project 1: Screening for new drug targets.

Project 2: Site-specific incorporation of L-acetyl-phenylalanine and L-benzoyl-phenylalanine into a target protein (X) in mammalian cells.

Grading

Successful conclusion of the course requires the completion of two projects. Attendance at all meetings is very important, and no more than one class can be missed. Students who miss a class must complete a make-up assignment, which will consist of a 1-2 page (double-spaced) summary of the papers discussed in that particular meeting.

Calendar

Course schedule.
SES #	TOPICS
1	Introduction
2	Toxins I: Toxins that target the ribosome - Ricin and Shiga toxin
3	Toxins II: Toxins that target eukaryotic elongation factor 2 - Diphtheria toxin and Pseudomonas exotoxin A
4	Toxins III: Toxins that target tRNA - Bacterial colicins and the eukaryotic γ-toxin
5	Mechanism and action of antibiotics I: Tetracycline and other antibiotics that target the 30S ribosomal subunit
6	Mechanism and action of antibiotics II: Chloramphenicol and other antibiotics that target the 50S ribosomal subunit
7	Mechanism and action of antibiotics III: Aminoglycoside antibiotics - Antibiotics that affect translational fidelity
8	Protein engineering I: Incorporation of unnatural amino acids into proteins - Making SENSE out of NON-SENSE
9	Protein engineering II: Incorporation of unnatural amino acids into proteins - Use of ribosomal frameshift-suppressor tRNAs and editing-defective aminoacyl-tRNA synthetases
10	Protein engineering III: In vitro evolution of proteins
11	The translational apparatus and human diseases
12	Epilogue

Readings

This section contains documents that could not be made accessible to screen reader software. A "#" symbol is used to denote such documents.

Help support MIT OpenCourseWare by shopping at Amazon.com! MIT OpenCourseWare offers direct links to Amazon.com to purchase the books cited in this course. Click on the Amazon logo to the left of any citation and purchase the book from Amazon.com, and MIT OpenCourseWare will receive up to 10% of all purchases you make. Your support will enable MIT to continue offering open access to MIT courses.

This section includes the readings that are required prior to the first class, a list of supplementary readings, and a list of readings required for each class session.

Required Readings (prior to start of course)

Prior to the first class, students should read:

Amazon logo Berg, Jeremy, John Tymoczko, and Lubert Stryer. Biochemistry. 5th ed. New York, NY: W.H. Freeman and Company, 2006, chapter 29. ISBN: 9780716787242.

These sections are well written, relatively short and give the essentials. The text is online, with links provided below:

29. Protein synthesis

29.1 Protein Synthesis Requires the Translation of Nucleotide Sequences Into Amino Acid Sequences

29.2 Aminoacyl-transfer RNA Synthetases Read the Genetic Code

29.3 A Ribosome Is a Ribonucleoprotein Particle (70S) Made of a Small (30S) and a Large (50S) Subunit

29.4 Protein Factors Play Key Roles in Protein Synthesis

29.5 Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation

Protein synthesis: Summary

Chapter 29: Protein Synthesis: Living Figure (requires Netscape® and Chime)

Supplementary Readings

In-depth information about the three-dimensional structure of the prokaryotic and eukaryotic ribosome.

Yusupov, M. M., G. Z. Yusupova, A. Baucom, K. Lieberman, T. N. Earnest, J. H. Cate, and H. F. Noller. "Crystal Structure of the Ribosome at 5.5 A Resolution." Science 292 (2001): 883-896.

Nissen, P., J. Hansen, N. Ban, P. B. Moore, and T. A. Steitz. "The Structural Basis of Ribosome Activity in Peptide Bond Synthesis." Science 289 (2000): 920-930.

Spahn, C. M., R. Beckmann, N. Eswar, P. A. Penczek, A. Sali, G. Blobel, and J. Frank. "Structure of the 80S Ribosome from Saccharomyces Cerevisiae: tRNA-ribosome and Subunit-subunit Interactions." Cell 107 (2001): 373-386.

Brodersen, D. E., W. M. Clemons Jr., A. P. Carter, R. J. Morgan-Warren, B. T. Wimberly, and V. Ramakrishnan. "The Structural Basis for the Action of the Antibiotics Tetracycline, Pactamycin, and Hygromycin B on the 30S Ribosomal Subunit." Cell 103 (2000): 1143-1154.

Weekly Readings by Session

Course readings.
SES #	TOPICS	READINGS	LINKS
1	Introduction	Biochemistry. 5th ed. Chapter 29. (See links above.)
2	Toxins I: Toxins that target the ribosome - Ricin and Shiga toxin	Gluck, A., and I. G. Wool. "Determination of the 28 S Ribosomal RNA Identity Element (G4319) for Alpha-sarcin and the Relationship of Recognition to the Selection of the Catalytic Site." J Mol Biol 256 (1996): 838-848. Reisbig, R., S. Olsnes, and K. Eiklid. "The Cytotoxic Activity of Shigella Toxin. Evidence for Catalytic Inactivation of the 60 S Ribosomal Subunit." J Biol Chem 256 (1981): 8739-8744.	In vitro transcription In vitro translation SDS-PAGE
3	Toxins II: Toxins that target eukaryotic elongation factor 2 - Diphtheria toxin and Pseudomonas exotoxin A	Iglewski, B. H., and D. Kabat. "NAD-dependent Inhibition of Protein Synthesis by Pseudomonas Aeruginosa Toxin." Proc Natl Acad Sci U.S.A. 72 (1975): 2284-2288. Kohno, K., and T. Uchida. "Highly Frequent Single Amino Acid Substitution in Mammalian Elongation Factor 2 (EF-2) Results in Expression of Resistance to EF-2-ADP-ribosylating Toxins." J Biol Chem 262 (1987): 12298-12305.	Two-dimensional gel electrophoresis Trypsin Diphtheria Pseudomonas
4	Toxins III: Toxins that target tRNA - Bacterial colicins and the eukaryotic γ-toxin	Tomita, K., T. Ogawa, T. Uozumi, K. Watanabe, and H. Masaki. "A Cytotoxic Ribonuclease which Specifically Cleaves Four Isoaccepting Arginine tRNAs at their Anticodon Loops." Proc Natl Acad Sci U.S.A. 97 (2000): 8278-8283. Lu, J., B. Huang, A. Esberg, M. J. O. Johansson, and A. S. Byström. "The Kluyveromyces Lactis γ -toxin Targets tRNA Anticodons." RNA 11 (2005): 1648-1654.	RNA sequencing (PDF - 1.3 MB) Primer extension (PDF)
5	Mechanism and action of antibiotics I: Tetracycline and other antibiotics that target the 30S ribosomal subunit	Moazed, D., and H. F. Noller. "Interaction of Antibiotics with Functional Sites in 16S Ribosomal RNA." Nature 327 (1987): 389-394. Wu, J. Y., J. J. Kim, R. Reddy, W. M. Wang, D. Y. Graham, and D. H. Kwon. "Tetracycline-resistant Clinical Helicobacter Pylori Isolates with and without Mutations in 16S rRNA-encoding Genes." Antimicrob Agents Chemother 49 (2005): 578-583. Extra reading Brodersen, D. E., W. M. Clemons Jr., A. P. Carter, R. J. Morgan-Warren, B. T. Wimberly, and V. Ramakrishnan. "The Structural Basis for the Action of the Antibiotics Tetracycline, Pactamycin, and Hygromycin B on the 30S Ribosomal Subunit." Cell 103 (2000): 1143-1154 and figure 2, page 1146.	RNA footprinting - enzymatic and chemical probes (PDF) Helicobacter pylori Bacteria of medical importance
6	Mechanism and action of antibiotics II: Chloramphenicol and other antibiotics that target the 50S ribosomal subunit	Thompson, J., D. F. Kim, M. O'Connor, K. R. Lieberman, M. A. Bayfield, S. T. Gregory, R. Green, H. F. Noller, and A. E. Dahlberg. "Analysis of Mutations at Residues A2451 and G2447 of 23S rRNA in the Peptidyltransferase Active Site of the 50S Ribosomal Subunit." Proc Natl Acad Sci U.S.A. 98 (2001): 9002-9007. Recht, M. I., S. Douthwaite, and J. D. Puglisi. "Basis for Prokaryotic Specificity of Action of Aminoglycoside Antibiotics." EMBO J 18 (1999): 3133-3138.
7	Mechanism and action of antibiotics III: Aminoglycoside antibiotics - Antibiotics that affect translational fidelity	Howard, M. T., C. B. Anderson, U. Fass, S. Khatri, R. F. Gesteland, J. F. Atkins, and K. M. Flanigan. "Readthrough of Dystrophin Stop Codon Mutations Induced by Aminoglycosides." Ann Neurol 55 (2004): 422-426. Salas-Marco, J., and D. M. Bedwell. "Discrimination Between Defects in Elongation Fidelity and Termination Efficiency Provides Mechanistic Insights into Translational Readthrough." J Mol Biol 348 (2005): 801-815.	Muscular dystrophy Muscular dystrophy
8	Protein engineering I: Incorporation of unnatural amino acids into proteins - Making SENSE out of NON-SENSE	Bain, J. D., C. Switzer, A. R. Chamberlin, and S. A. Benner. "Ribosome-mediated Incorporation of a Non-standard Amino Acid into a Peptide through Expansion of the Genetic Code." Nature 356 (1992): 537-539. Chin, J. W., T. A. Cropp, J. C. Anderson, M. Mukherji, Z. Zhang, and P. G. Schultz. "An Expanded Eukaryotic Genetic Code." Science 301 (2003): 964-967.	Protein engineering (PDF)
9	Protein engineering II: Incorporation of unnatural amino acids into proteins - Use of ribosomal frameshift-suppressor tRNAs and editing-defective aminoacyl-tRNA synthetases	Hohsaka, T., Y. Ashizuka, H. Taira, H. Murakami, and M. Sisido. "Incorporation of Non-natural Amino Acids into Proteins by Using Various Four-base Codons in an Escherichia Coli in Vitro Translation System." Biochemistry 40 (2001): 11060-11064. Döring, V., H. D. Mootz, L. A. Nangle, T. L. Hendrickson, V. de Crécy-Lagard, P. Schimmel, and P. Marlière. "Enlarging the Amino Acid Set of Escherichia Coli by Infiltration of the Valine Coding Pathway." Science 292 (2001): 501-504.
10	Protein engineering III: In vitro evolution of proteins	Roberts, R. W., and J. Szostak. "RNA-peptide Fusions for in Vitro Selection of Peptides and Proteins." Proc Natl Acad Sci U.S.A. 94 (1997): 12297-12302. Jermutus, L., A. Honegger, F. Schwesinger, J. Hanes, and A. Plückthun. "Tailoring in Vitro Evolution for Protein Affinity or Stability." Proc Natl Acad Sci U.S.A. 98 (2001): 75-80.
11	The translational apparatus and human diseases	Kirino, Y., T. Yasukawa, S. Ohta, S. Akira, K. Ishihara, K. Watanabe, and T. Suzuki. "Codon-specific Translational Defect Caused by a Wobble Modification Deficiency in Mutant tRNA from a Human Mitochondrial Disease." Proc Natl Acad Sci U.S.A. 101 (2004): 15070-15075. Lee, J. W., K. Beebe, L. A. Nangle, J. Jang, C. M. Longo-Guess, S. A. Cook, M. T. Davisson, J. P. Sundberg, P. Schimmel, and S. L. Ackerman. "Editing-defective tRNA Synthetase Causes Protein Misfolding and Neurodegeneration." Nature 443 (2006): 50-55. Sako, Y., F. Usuki, and H. Suga. "A Novel Therapeutic Approach for Genetic Diseases by Introduction of Suppressor tRNA." Nucleic Acids Symp Ser 50 (2006): 239-240.
12	Epilogue	Oral presentations and evaluation of 2nd assignments. General discussion: "The Protein Primer" Movie by Paul Berg who won The Nobel Prize in Chemistry in 1980. On an open field at Stanford University in 1971, several hundred students convened to undulate and impersonate molecules undergoing protein synthesis by a ribosome. A few were trained dancers, wearing costumes and colored balloons to identify their roles; most were recruited with the promise of fun and refreshments. But make no mistake: despite the flower-power feel and psychedelic strains of the "Protein Jive Sutra," this is serious science. The narrator is Nobel laureate Paul Berg, who explains the process in a prologue that introduces the leading players, such as 30s Ribosome, mRNA, and Initiator Factor One.