Teaching Argumentation and Scientific Discourse Using the Ribosomal Peptidyl Transferase Reaction &s

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1 Q 2011 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 3, pp , 2011 Articles Teaching Argumentation and Scientific Discourse Using the Ribosomal Peptidyl Transferase Reaction &s Received for publication, October 5, 2010, and in revised form, November 30, 2010 R. Jeremy Johnson From the Department of Chemistry, Butler University, 4600 Sunset Ave, Indianapolis, IN Argumentation and discourse are two integral parts of scientific investigation that are often overlooked in undergraduate science education. To address this limitation, the story of peptide bond formation by the ribosome can be used to illustrate the importance of evidence, claims, arguments, and counterarguments in scientific discourse. With the determination of the first structure of the large ribosomal subunit bound to a transition state inhibitor came an initial hypothesis about the role of the ribosome in peptide bond formation. This initial hypothesis was based on a few central assumptions about the transition state mimic and acid base catalysis by serine proteases. The initial proposed mechanism started a flurry of scientific discourse in experimental articles and commentaries that tested the validity of the initial proposed mechanism. Using this civil argumentation as a guide, class discussions, assignments, and a debate were designed that allow students to analyze and question the claims and evidence about the mechanism of peptide bond synthesis. In the end, students develop a sense of critical skepticism, and an understanding of scientific discourse, while learning about the current consensus mechanism for peptide bond synthesis. Keywords: Peptide bond synthesis, ribosome, scientific discourse, critical skepticism. Peptide bond synthesis by the ribosome is an essential chemical reaction to life, but until the three-dimensional structure of the ribosome was determined, only a basic outline of the reaction catalyzed by the ribosome was known (Fig. 1A) [1]. Francis Crick had hypothesized in 1968 that the ribosome was actually a ribozyme, but until 2000, only limited evidence supported this theory [2]. The structure of the ribosome solidified the assertion that the ribosome was a ribozyme, as no amino acid lay closer than 17 Å to the site of peptidyl transfer (Figs. 1C and 1D) [1, 3]. The question then became how RNA could accomplish peptide bond synthesis, and the structure of the large ribosomal subunit bound to a transition state mimic ignited a flurry of activity to provide a detailed molecular explanation of the peptidyl transferase reaction (Fig. 1). With its central role in molecular science and the defining accomplishment of determining its threedimensional structure, understanding the peptidyl transferase reaction catalyzed by the ribosome provides an &s Additional supporting Information may be found in the online version of this article. To whom correspondence should be addressed. Butler University, 4600 Sunset Ave, Indianapolis, Indiana Tel: rjjohns1@butler.edu. This paper is available on line at exciting example of scientific experimentation and discovery [1, 4]. The vibrant debate that the initial mechanism started also illustrates the role that scientific discourse and argumentation play in scientific investigation. In the end, understanding the structure of the ribosome and the mechanism of peptide bond synthesis proved problems deserving of the 2009 Nobel Prize in Chemistry [4]. PEDAGOGICAL IMPORTANCE Argumentation and active discourse are two integral parts of scientific investigation that are often overlooked in undergraduate scientific education, but a growing area of study on scientific education shows that students learn better through critical, collaborative discourse [5]. These studies emphasize the need for students to practice identifying scientific claims, reasons, evidence, and counterarguments, and that this practice can be accomplished by helping students see how that knowledge came to be accepted [5, 6]. To begin to teach scientific discourse, Alberts argues that undergraduate science education needs to stress the importance of the scientific process instead of teaching students only about what scientists know [7]. The ribosomal peptidyl transferase reaction provides a perfect story for emphasizing the process of scientific discovery, the evidential nature of scientific argumenta- DOI /bmb.20495

2 186 BAMBED, Vol. 39, No. 3, pp , 2011 FIG. 1. Basic peptidyl transferase mechanism and ribosomal structure. A: Basic peptidyl transferase reaction. The amino group on the A-site aminoacylated-trna attacks the carbonyl on the P-site bound peptidyl-trna. Product formation proceeds through a tetrahedral intermediate. B: Proposed peptidyl transferase reaction based on the structure of CCdAp-Puromycin bound to the large ribosomal subunit [1]. C: Large ribosomal subunit from Haloarcula marismortui shown (PDB accession code 1JJ2) with the peptidyl transferase center highlighted by CCdAp-Puromycin (PDB accession code 1FFZ) [1]. Puromycin depicted in black, 23S rrna in tan, 5S rrna in light green, and ribosomal proteins in blue. D: Large ribosomal subunit without rrna illustrates the distance between the peptidyl transferase center and the closest amino acid residue [1]. E: Close-up picture of the peptidyl transferase center without rrna. Figures A B constructed in ChemDraw (CambridgeSoft). Figures C E made using PyMol (Schršdinger LLC). tion, and the development of critical skepticism. To introduce scientific argumentation and discourse, an upper level undergraduate biochemistry course is led through a 1- to 2-week module of classroom discussion, assignments, and a debate that traces the scientific understanding of peptide bond formation by the ribosome. This module is part of a lecture presentation on the central dogma of biochemistry that is given as part of the second semester of a two-semester biochemistry course. The students in the class are senior or junior chemistry or biology majors that have received a C or better in the first semester of biochemistry. Students are assigned six journal articles to study (Table I) and are given basic questions to answer while reading to help with their comprehension. An active classroom discussion is then facilitated where students questions and responses are expected to drive the conversation TABLE I Journal articles used to trace the understanding of peptide bond synthesis by the ribosome Title of the journal article Citation 1) The structural basis of ribosome activity in peptide bond synthesis 1 2) Mechanism of ribosomal peptide bond formation 8 3) Evidence against stabilization of the transition state oxyanion 9 by a pk a -perturbed RNA base in the peptidyl transferase center 4) The ribosome as an entropy trap 10 5) Structural insights into the roles of water and the 2 0 hydroxyl of the P-site trna 11 in the peptidyl transferase reaction 6) A structural view on the mechanism of the ribosome-catalyzed peptide bond formation 12

3 TABLE 2 Outline of classroom instruction Classroom activities Module day A) Assign reading assignments (Table I). 1 B) Give an introduction to peptidyl transfer, describe the overall goal of the module, and discuss 8 the Nobel Prize awarded for the structure of the ribosome. Collect student written questions. Assign students to three groups for the classroom debate. C) Discuss the structure of the ribosome, the evidence that the ribosome is a ribozyme, 9 and the crystallization of the ribosome. Describe the proposed mechanism of peptide bond synthesis. Give the students time in small groups to find the evidence supporting the initial mechanism and then make a list of the evidence. D) Go through the important assumptions made in proposing the initial mechanism, 10 including the relevance of CCdAp-Puromycin as a transition state mimic, the missing 2 0 OH on CCdAp-Puromycin, and the shifted pk a of N3 on A2486. Point out the proposed tautomerization of N3 on A2486. E) Lead a classroom debate on the mechanism of peptide bond synthesis. 11 Focus the discussion on the shifted pk a of N3 on A2486, the missing 2 0 OH on CCdAp-Puromycin, and modeling peptidyl transfer on amide bond hydrolysis by serine proteases. F) Examine the three papers [8 10] contradicting the proposed mechanism. 12 Assign different figures from each paper to a small group of students. Have them give a short presentation explaining the figure and the important conclusions from the figure. G) Continue discussion on the three papers, focusing on the relevance of 13 CCdAp-Puromycin as a transition state mimic, the experimental methods used to disprove the initial mechanism, and changes to the initial mechanism. H) Wrap up the discussion by having students examine the current consensus mechanism. 14 Have them explain how the ribosome catalyzes peptide bond formation and how this mechanism changed from the initial proposed mechanism. I) Edit student designed test questions and return for exam preparation (Table II). The discussion is continually steered to help students focus on the main evidence and arguments in each paper and to follow the logical order of the experimental findings. In the end, students are expected to synthesize into a complete argument the problems with the initial hypothesis about the peptidyl transferase reaction and how new experimental evidence refined the initial mechanism. Student comprehension is then assessed by short writing assignments and examination questions that are written by the students (Supporting Information). The goal of this 1- to 2-week module is to challenge the students understanding of scientific knowledge, to expose them to an active area of scientific discourse, and to involve them in the critical evaluation of scientific evidence and claims. INITIAL DISCOVERY AND HYPOTHESES The first molecular picture of the peptidyl transferase reaction emerged from the crystal structure of the large ribosomal subunit from Haloarcula marismortui in complex with two substrate analogs, CCdAp-Puromycin and an N-amino-acylated minihelix [1]. The N-amino-acylated minihelix provided only limited information about the reaction, but the CCdAp-Puromycin provided a molecular picture of the proposed transition state. CCdAp-Puromycin is a transition state analog where CCdA mimics the universal CCA sequence on the 3 0 -termini of all trnas, the phosphoramide linkage mimics the tetrahedral transition state, and Puromycin mimics A-site binding (Fig. 2) [1]. FIG. 2. CCdAp-Puromycin as a transition state mimic. A: CCdAp-Puromycin bound to the P-site and A-site in the large ribosomal subunit of H. marismortui [1]. One of the negatively charged oxygens of the phosphoramide linkage forms a hydrogen bond to Adenine 2486 in the 23S rrna and is proposed to stabilize the negative charge on the tetrahedral transition state [1]. Figure made using PyMol (Schršdinger LLC). B: Chemical structure of CCdAp-Puromycin that highlights the phosphoramide linkage and the missing 2 0 OH on the adenine residue that mimics A76 of the aminoacyl-trna [1, 8]. The chemical structure of CCdAp-Puromycin is drawn to mirror the structure of CCdAp-Puromycin bound to the ribosome (Fig. 2A) and the ribose sugars are numbered to highlight the location of the missing 2 0 OH. Figure made using ChemDraw (CambridgeSoft).

4 188 BAMBED, Vol. 39, No. 3, pp , 2011 FIG. 3. Investigating the peptidyl transferase reaction. A: Proposed tautomeric interconversion of G2482 and A2486 in the 23S rrna caused by a buried phosphate at A2485 [1]. B: Simple mimics of the peptidyl transferase reaction used to determine the uncatalyzed rate of peptide bond formation [9]. C: Consensus peptidyl transferase reaction where the 2 0 OH of A76 orients the amino group and shuttles protons in the transition state and where a tightly bound water molecule stabilizes the tetrahedral transition state [11]. Figures made using ChemDraw (CambridgeSoft). Based on the proximity of the phosphoramide oxygen of CCdAp-Puromycin to N3 of A2486 in the 23S rrna, the peptidyl transferase reaction was hypothesized to be an acid base reaction catalyzed by N3 of A2486 (Figs. 1B and 2) [1]. Adenine 2486 is proposed to act as a base to remove a proton from the nucleophilic amino group on the amino-acylated trna and to act as an acid to protonate the 3 0 OH on the A-site trna (Fig. 1D). For A2486 to act as a general acid base and to protonate the trna leaving group, the pk a value of N3, which is normally about 1.5 (indicating that N3 would not readily act as a general base), must shift approximately 5.5 units higher to around 7 [1]. Evidence for this abnormal pk a shift is provided by the observation that A2486 is protonated at ph 5.8 (the ph of the ribosomal crystal). This shift is proposed to arise from a buried phosphate at A2485 that could lead to a tautomeric shift of G2482 and A2486 (Fig. 3A). This would raise the pk a value of N3 in the rare A2486 tautomer, as the proposed negative charge on the N3 nitrogen causes it to be more basic [1]. The initially proposed peptidyl transferase mechanism was predicated on a couple important assumptions. First, one of the negatively charged oxygens on the phosphoramide group was assumed to be oriented in the same direction as the negatively charged oxygen on the tetrahedral intermediate. Second, the missing 2 0 OH on CCdAp-Puromycin was assumed to be irrelevant to the peptidyl transferase reaction. Picking out these important assumptions can be difficult for the students, but identifying and assessing the validity of these assumptions is necessary for future classroom discussion about the proposed mechanism. To help them identify the main points while reading, the students are instructed to describe the hypothesized mechanism for peptidyl transferase first, to find the evidence used to support the mechanism, and to decide if they are convinced by the evidence. In-class discussion then focuses on the evidence to support the mechanism and tries to challenge students to question the evidence presented. From this initial mechanism of peptidyl transfer involving acid base catalysis by A2486, starts the process of model refinement and scientific discourse. OPEN DEBATE What makes the story of the peptidyl transferase reaction especially relevant for discussing argumentation and discourse in science is a short technical comment entitled, Mechanism of ribosomal peptide bond formation that appeared in Science magazine five months after the initial structure [8]. In this technical comment, two alternative explanations for peptide bond synthesis by the ribosome were proposed along with counterarguments from the authors of the original mechanism. The alternative mechanisms questioned the structural relevance of CCdAp-Puromycin as a transition state mimic, the role of A2486 in catalysis, the importance of the missing 2 0 OH on CCdAp- Puromycin, and the highly shifted pk a value of A2486 [8]. The main argument revolved around the deprotonation of the amino group on the aminoacyl-trna by A2486 and provides an excellent opportunity to discuss deprotonation and pk a values in enzyme mechanisms. The original authors then defended their mechanistic assertions and expanded the link between their peptidyl transferase reaction and the acylation reaction of serine proteases [8]. The technical comments read like a debate where logical arguments, evidence, and claims are presented and discussed. To teach this section, the classroom discussion is set up as a debate, where students are separated into small groups and assigned one of the sections in the

5 189 technical comment (Supporting Information). Classroom discussion begins with one group presenting contradictory evidence against the original reaction mechanism and the other groups then giving counterarguments. Once students have carefully examined the evidence presented in the technical comment, the discussion of future experimental studies and their results match up with the student s own growing skepticism about the original proposed mechanism (Fig. 1B). EXPERIMENTAL DISCOURSE Since 2000 when the initial mechanism was proposed, the current model for peptide bond synthesis has been refined through continual cycles of discourse and experimentation. For classroom discussion, three experimental articles were chosen that challenged the original mechanism using the results of different experimental techniques (Table I) [9 11]. While reading each article, students are instructed to focus on the main hypothesis, on the connection to the original peptidyl transferase mechanism, and on the proposed changes to the reaction mechanism (Table II). Evidence against pk a Stabilization The first article describes a simple experiment that tests the existence of the proposed pk a shift of N3 in A2486 by measuring the ph dependence of the binding of CCdAp-Puromycin to ribosomes from ph 5.0 to 8.5 [9]. CCdAp-Puromycin is a mimic of the transition state and this proposed transition state is stabilized by a hydrogen bond to N3 of A2486. Thus, the protonation or deprotonation of this nucleotide with ph should change its hydrogen bonding ability and, thus, its affinity for binding to CCdAp-Puromycin. Instead of finding the ph variability that would have been expected given the original mechanism, CCdAp-Puromycin binding is invariant from ph 5.0 to 8.5 [9]. Additionally, these experiments examine the quality of CCdAp-Puromycin as a mimic of the peptidyl transferase reaction and assists students in developing their skepticism of CCdAp-Puromycin. The simple experiment also shows how the knowledge taught in an undergraduate biochemistry class can directly address an important scientific question. Ribosome as an Entropy Trap The second article addresses the role of the ribosome in peptidyl transfer from a different angle and probes the thermodynamics (heats and entropies of activation) of the peptidyl transferase reaction using chemical mimics of peptide bond formation (Fig. 3B) [10]. NMR spectroscopy was used to compare the temperature dependence of the catalyzed and uncatalyzed reactions and the heats and entropies of activation were calculated using the Arrhenius plot. Based on the activation energies, the ribosome-catalyzed reaction was calculated to accelerate peptidyl transfer by fold, and the entire rate increase is attributed to the lowering of the entropy of activation [10]. Although this second paper does not directly test the original mechanism for peptide bond synthesis, it provides an alternate view of the role of the ribosome where the ribosome serves to preorient the substrates. This explanation of the peptidyl transferase reaction proves to be complementary to the active participation of the ribosome in peptide bond synthesis and challenges students to combine multiple arguments into a concerted final mechanism. Transition State Stabilization Revisited The third paper answers two important questions from the original mechanism: did CCdAp-Puromycin accurately mimic the tetrahedral transition state of the peptidyl transferase reaction and what was the role of the 2 0 OH of the peptidyl-trna? [11] Crystal structures with new mimics show that the negatively charged phosphoramide oxygen of CCdAp-Puromycin was directed away from the true oxyanion hole, where now a bound water molecule stabilizes the tetrahedral transition state [11]. The 2 0 OH of the peptidyl-trna is also found to have an active role in catalysis where it hydrogen bonds to the nucleophilic amino group and shuttles protons between the amino group and the 3 0 OH of A76 of the peptidyl-trna [11]. This paper addresses all of the previous arguments raised in the technical comment and research papers and provides answers to the points of disagreement. These experiments and explanations dismiss the role of A2486 in the peptidyl transferase reaction and remove any necessity to discuss pk a shifts in buried nucleotides. It also provides another chance to teach about models and assumptions, and how the new transition state analogs are improved over CCdAp-Puromycin. CURRENT CONSENSUS MECHANISM Although active research on the peptidyl transferase reaction is still proceeding, a consensus has been reached about the role of the ribosome in the peptidyl transferase reaction [12]. This consensus provides a satisfying conclusion to the years of discussion and experimentation and can be clearly illustrated in a single reaction diagram (Fig. 3C). The final mechanism highlights the important role of the 2 0 OH on A76 in orienting the amino group of aminoacyl trna and in proton shuttling. It also shows the role of water in stabilizing the negative charge on the tetrahedral transition state [12]. This representation cannot directly show the role of substrate preorganization in the activity of the ribosome, but it is indirectly stated through the proper orientation of the amino group on the aminoacyl-trna and the carbonyl on the peptidyl-trna. For discussing the updated mechanism, a recent review of the structural understanding of the peptidyl transferase reaction is read and discussed [12]. Students are instructed to focus on the subsection of the paper describing the consensus mechanism and to construct two exam questions related to the structure and mechanism (Supporting Information). Constructing the exam questions leads students to consider the current mechanism critically and what questions challenged their own understanding of the mechanism and structure.

6 190 BAMBED, Vol. 39, No. 3, pp , 2011 CONCLUSIONS The three-dimensional structure of the 50S ribosome bound to CCdAp-Puromycin provided a testable hypothesis about the peptidyl transferase reaction that involved acid base catalysis and transition state stabilization (Figs. 1 and 2) [1]. This initial mechanism precipitated a lively discussion that over 10 years and through multiple revisions led to a consensus understanding of peptide bond formation by the ribosome and the awarding of the 2009 Nobel Prize in Chemistry (Fig. 3C) [1, 8 11]. The story of the ribosomal peptidyl transferase mechanism illustrates the role that scientific discourse and argumentation play in steering scientific understanding toward a consensus [5 7]. Through the classroom discussion of this story, students are challenged to comprehend complex scientific journal articles, to identify the evidence and assumptions, and to question published scientific findings (Supporting Information). The reading assignments also reiterate important topics in molecular life science such as protein/ nucleic acid structure, enzyme rate enhancement, acid base catalysis, and nucleophilicity. Once students grasp the initial mechanism and are led to question its relevance, the debate surrounding the mechanism excites their curiosity and challenges their view of scientific progression. In the end, the module provides a new picture of how scientific understanding is gained and how critical skepticism and discourse shape scientific knowledge. REFERENCES [1] P. Nissen, J. Hansen, N. Ban, P. B. Moore, T. A. Steitz (2000) The structural basis of ribosome activity in peptide bond synthesis, Science. 289, [2] F. H. C. Crick (1968) The origin of the genetic code, J. Mol. Biol. 38, [3] W. A. Decatur ( 2010) Proteopedia entry: The large ribosomal subunit of Haloarcula marismortui, Biochem. Mol. Biol. Educ. 38, 343. [4] R. F. Service ( 2009) Chemistry Nobel. Honors to researchers who probed atomic structure of ribosome, Science. 326, [5] J. Osborne (2010) Arguing to learn in science: The role of collaborative, critical discourse, Science. 328, [6] J. S. Krajcik, L. M. Sutherland ( 2010) Supporting students in developing literacy in science, Science. 328, [7] B. Alberts ( 2009) Redefining science education, Science. 323, 437. [8] A. Barta, S. Dorner, N. Polacek, J. M. Berg, J. R. Lorsch, P. Nissen, J. Hansen, G. W. Muth, N. Ban, P. B. Moore, S. A. Strobel, T. A. Steitz ( 2001) Mechanism of ribosomal peptide bond formation, Science. 291, 203a. [9] K. M. Parnell, A. C. Seila, S. A. Strobel ( 2002) Evidence against stabilization of the transition state oxyanion by a pka-perturbed RNA base in the peptidyl transferase center. Proc. Natl. Acad. Sci. USA. 99, [10] A. Sievers, M. Beringer, M. V. Rodnina, R. Wolfenden (2004) The ribosome as an entropy trap, Proc. Natl. Acad. Sci. USA. 101, [11] T. M. Schmeing, K. S. Huang, D. E. Kitchen, S. A. Strobel, T. A. Steitz (2005) Structural insights into the roles of water and the 2 0 hydroxyl of the P site trna in the peptidyl transferase reaction, Mol. Cell. 20, [12] M. Simonović, T. A. Steitz (2009) A structural view on the mechanism of the ribosome-catalyzed peptide bond formation, Biochim. Biophys. Acta. 1789,