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7.344 | Spring 2007 | Undergraduate

# Antibiotics, Toxins, and Protein Engineering

### Required Readings (prior to start of course)

Prior to the first class, students should read:

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

### 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

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. |

## Course Info

##### Instructors

- Dr. Caroline Koehrer
- Dr. Mandana Sassanfar

##### Departments

- Biology

##### As Taught In

Spring 2007

##### Level

Undergraduate

##### Topics

- Engineering
  - Biological Engineering
- Health and Medicine
  - Pharmacology and Toxicology

##### Learning Resource Types

notes Lecture Notes
assignment Written Assignments

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