Problem-solving Test: The Mechanism of Protein Synthesis

Q 2009 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 37, No. 1, pp. 58 62, 2009 Problem-based Learning Problem-solving Test: The Mechanism of Protein Synthesis Received for publication, August 25, 2008, and in revised form, September 9, 2008 József Szeberényi* From the Department of Medical Biology, Medical School, University of Pécs, H-7624 Pécs, Hungary Terms to be familiar with before you start to solve the test: protein synthesis, ribosomes, amino acids, peptides, peptide bond, polypeptide chain, N- and C-terminus, hemoglobin, a- and b-globin chains, radioactive labeling, [ 3 H] and [ 14 C]leucine, cytosol, differential centrifugation, density gradient centrifugation, trypsin, electrophoresis, chromatography THE EXPERIMENT One of the most important studies of early molecular biology was performed by Howard Dintzis to analyze the mechanism of protein synthesis [1]. At that time very little was known of how proteins were manufactured: ribosomes had already been identified as sites of protein synthesis, but neither the template (mrna), nor the adapter function of trna molecules had been discovered yet. (A reflective article by the scientist about his discovery was recently published in BAMBED [2], from which we could learn, among many other things, that the term ribosome was actually introduced by him into the terminology of molecular biology.) The elegant experiments of Dintzis were designed to study the basic mechanism of polypeptide chain growth. Theoretically, several distinct mechanisms could have been imagined, the most plausible possibilities are shown in Fig. 1: (a) the amino acids line up along a putative template molecule and an enzyme catalyzes simultaneous peptide bond formation between them; (b) short oligopeptides are synthesized on the template and then ligated to form the polypeptide chain; (c) the polypeptide is synthesized by sequential addition of amino acids to the C-terminus of the growing chain; (d) continuous synthesis in the opposite direction. The work of Dintzis addressed this problem using immature rabbit red blood cells called reticulocytes. In these cells, 90% of proteins synthesized is hemoglobin. Newly synthesized globin chains are readily labeled in cultured reticulocytes using radioactive amino acids. The best choice for labeling hemoglobin turned out to be radioactive leucine. There are several important reasons for that. First, leucine is the most abundant amino acid in globin chains. 1. What is the advantage of this fact? A. Newly synthesized globin chains can be labeled to a high specific activity. B. Various regions of globin chains can be labeled. C. No other proteins will become radioactive. D. A and B. E. A, B and C. The second advantage of leucine is that it is an essential amino acid. 2. Why does this fact have an impact on protein labeling? A. Radioactive leucine is not diluted out by nonradioactive leucine molecules produced by the cell from other compounds. B. Radioactive leucine is not diluted out by nonradioactive leucine molecules stored in the cell. C. Radioactive leucine is rapidly incorporated into proteins before being converted into other molecules. D. A and C. E. A, B and C. In this study, reticulocyte cultures were labeled with [ 3 H]leucine for various periods of time. At the same time, other reticulocyte populations received a prolonged labeling with [ 14 C]leucine. The two cultures of cells were mixed and ribosomal and cytosolic fractions were prepared from them. * To whom correspondence should be addressed. Tel.: 36-72- 536216; Fax: 36-72-536453. E-mail: jozsef.szeberenyi@aok.pte.hu. DOI 10.1002/bmb.20239 58 This paper is available on line at http://www.bambed.org

59 FIG. 1. Hypothetical models of protein chain growth. (a) Simultaneous peptide bond formation between all adjacent amino acids, (b) synthesis and subsequent ligation of short peptides, unidirectional chain growth by serial addition of amino acids to the (c) C-terminus, or (d) N-terminus of the elongating polypeptide chain. (The N- and C-terminus of the polypeptide chain are indicated; small boxes correspond to individual amino acids). 3. What is the best way to isolate ribosomes and cytosol? A. Ultracentrifugation of homogenates. B. Ultracentrifugation of the postnuclear supernatant. C. Ultracentrifugation of the postmitochondrial supernatant. D. Cesium chloride gradient centrifugation of the homogenate. E. Sucrose gradient centrifugation of the homogenate. FIG. 2.Two-dimensional separation ( fingerprint analysis ) of tryptic digests of rabbit a-globin chains labeled with radioactive leucine. The fingerprint shows radioactively labeled oligopeptides only (for details see the text). a- and b-globin chains were isolated and purified from the ribosomal and cytosolic fractions and subjected to fingerprint analysis : proteins were digested with trypsin (a proteolytic enzyme cutting peptide bonds next to arginine and lysine) generating a set of peptides that were subsequently fractionated using a two-dimensional separation technique of electrophoresis and chromatography. Figure 2 shows such a peptide map of tryptic digests of radioactively labeled a-globin (peptides containing radioactive leucine are only shown). Fingerprint analysis of labeled soluble (cytosolic) a-globin molecules were performed after different durations of labeling (4, 7, and 60 minutes), the 3 H and 14 C radioactivities of the spots shown in Fig. 2 were determined, 3 H/ 14 C ratios were calculated for each spot and the peptides were ordered in increasing 3 H/ 14 C ratios (see Fig. 3a). A similar analysis was performed on ribosome-bound globin chains (Fig. 3b). FIG. 3.Distribution of [ 3 H]leucine among tryptic peptides of soluble (a) and ribosome-bound (b) rabbit a-globin chains (for experimental detail see the text). (Note: Analysis of newly synthesized, ribosome-bound proteins is a technically difficult task. Diagram B shows, for didactic reasons, an idealized interpretation of the results. For the actual data, please refer to the original paper [1].)

60 BAMBED, Vol. 37, No. 1, pp. 58 62, 2009 4. What was the aim of the double-labeling? A. To identify newly synthesized regions in [ 3 H]leucine pulse-labeled globin chains. B. To obtain uniformly labeled [ 14 C] globin chains. C. To use 14 C-radioactivity for each peptide as a measure of leucine content. D. To use 3 H/ 14 C ratios for each spot as a measure of relative 3 H labeling. E. All four statements are true. The hypothetical mechanisms shown in Fig. 1 would give different kinetics of 3 H globin labeling in the experiments described earlier. Let s try to predict these patterns! FIVE-CHOICE ASSOCIATION (This type of question consists of a list of lettered headings followed by a list of numbered words or phrases. For each numbered word or phrase, select the one heading which is most closely related to it.) A. Uniform or nearly uniform labeling of all leucine-containing B. Randomly uneven labeling of all leucine-containing C. Preferential labeling of C-terminal leucine-containing D. Preferential labeling of N-terminal leucine-containing E. None of the above patterns can be expected. 5. Mechanism A would give this pattern of labeling of ribosome-bound a-chains after brief exposure 6. Mechanism A would give this pattern of labeling of ribosome-bound a-chains after long exposure 7. Mechanism A would give this pattern of labeling of cytosolic a-chains after brief exposure 8. Mechanism A would give this pattern of labeling of cytosolic a-chains after long exposure 9. Mechanism B would give this pattern of labeling of ribosome-bound a-chains after brief exposure 10. Mechanism B would give this pattern of exposure 11. Mechanism B would give this pattern of 12. Mechanism B would give this pattern of labeling of cytosolic a-chains after long exposure 13. Mechanism C would give this pattern of labeling of ribosome-bound a-chains after brief exposure 14. Mechanism C would give this pattern of exposure 15. Mechanism C would give this pattern of 16. Mechanism C would give this pattern of labeling of cytosolic a-chains after long exposure 17. Mechanism D would give this pattern of labeling of ribosome-bound a-chains after brief exposure 18. Mechanism D would give this pattern of exposure 19. Mechanism D would give this pattern of 20. Mechanism D would give this pattern of labeling of cytosolic a-chains after long exposure If you answered the above set of questions correctly and you analyzed the curves of Fig. 3 carefully, you already know that a single question is left to be answered to decide which of the four mechanisms is true for globin synthesis. 21. What can be this question? A. Which labeled peptide contains the most leucine residues? B. Which labeled peptides contain only one leucine residue? C. Which labeled peptide is closest to one end of a-globin? D. Which labeled peptide has the strongest charge? E. Which labeled peptide has the highest molecular weight? To answer this question, a-globin uniformly labeled with [ 14 C]leucine was briefly digested with carboxypeptidase, an enzyme that removes amino acids from the C-terminus of proteins. The digested a-globin was then mixed with intact, uniformly [ 3 H]leucine-labeled a-chains and the mixture was subjected to tryptic fingerprinting. 3 H- and 14 C- radioactivities were measured in each peptide spot. Table I shows the 3 H/ 14 C ratios for all labeled TABLE I 3 H/ 14 C ratios in the tryptic peptides of untreated [ 3 H]leucine-labeled and carboxypeptidase-digested [ 14 C]-labeled rabbit (-globin chains (for details see the text) Peptide number 3 H/ 14 C ratio 10 1.0 11 1.0 14 1.0 16 11.0 20 1.0 21 1.0 22 0.9 25 1.0 31 1.0

61 FIG. 4. Model of sequential chain growth. Horizontal lines represent unlabeled polypeptide regions, horizontal boxes indicate [ 3 H]leucine-labeled sections. Horizontal arrows indicate the release of finished polypeptide chains from the ribosomes to the cytosol. Further details are described in the text. 22. What conclusion can be drawn from the carboxypeptidase experiment? A. The N-terminal amino acid of rabbit a-globin is arginine or lysine. B. The C-terminal amino acid of rabbit a-globin is arginine or lysine. C. Peptide 16 gives the C-terminus of rabbit a- globin. D. Peptide 16 gives the N-terminus of rabbit a- globin. E. Peptide 16 is closest to the C-terminus among the radioactively labeled tryptic 23. We can now describe the exact mechanism of protein synthesis. a-globin chains are synthesized. A. By simultaneous enzymatic ligation of adjacent amino acids. B. By simultaneous enzymatic ligation of preformed short C. By adding amino acids one at a time to the growing polypeptide chain in N- to C-terminal direction. D. By adding amino acids one at a time to the growing polypeptide chain in C- to N-terminal direction. E. By a mechanism different from the above described mechanisms. CORRECT ANSWERS 1. D 2. D 3. C 4. E 5. A 6. A 7. A 8. A 9. A 10. A 11. A 12. A 13. A 14. D 15. C 16. A 17. A 18. C 19. D 20. A 21. C 22. E 23. C EXPLANATIONS The experiment presented in this test was designed to analyze the sequential nature and direction of protein synthesis. Pulse-labeling with an abundant amino acid, leucine, provided the possibility to identify different regions of the protein and to determine the order of their labeling during protein synthesis (MCQ 1: D; MCQ 2: D). 1 The double-labeling protocol gave a chance to determine the fraction of newly synthesized (i.e. [ 3 H]leucine pulse-labeled) peptides in each tryptic fingerprint spot (MCQ 4: E). Simultaneous joining of amino acids (mechanism (a) in Fig. 1) or preformed peptides (mechanism (b)) would have given uniform labeling of nascent (ribosome-bound) or finished (cytosolic) globin chains, no matter what the duration of labeling was (MCQ 5 to 12: A). Sequential, unidirectional synthesis would radioactively label the elongating end of the polypeptide chain first. Fig. 4 helps to interpret the results. 1 The abbreviation used is: MCQ, multiple-choice question.

62 BAMBED, Vol. 37, No. 1, pp. 58 62, 2009 At the time of adding [ 3 H]leucine to the cells (0 minutes in Fig. 4) ribosomes carry nascent globin chains of various length. Brief labeling gives short radioactive sections to the growing end of each globin chain that will give a uniform labeling to all peptides (MCQ 13: A; MCQ 17: A). Completed chains are released to the cytosol and, after very short exposure to [ 3 H]leucine, their recently finished ends are only labeled (MCQ 15: C; MCQ 19: D). After long labeling more and more uniformly labeled globin chains are released from the ribosomes, the gradient of labeling between the two ends decreases and finally disappears (MCQ 16: A; MCQ 20: A). In contrast, a gradient of radioactivity is formed after long-term labeling on ribosome-bound, nascent protein chains from the initial to the final peptide (see Fig. 4, MCQ 14: D; MCQ 18: C). Since the carboxypeptidase experiment proved that peptide 16 was closest to the C-terminus (MCQ 21: C; MCQ 22: E), the data presented in this test strongly supports the hypothesis that translation continuously proceeds from the N- to the C-terminus of the polypeptide chain (MCQ 23: C). The best way to isolate ribosomal and cytosolic fractions is differential centrifugation of cell homogenates (MCQ 3: C). REFERENCES [1] H. M. Dintzis (1961) Assembly of the peptide chains of hemoglobin, Proc. Natl. Acad. Sci. USA 47, 247 261. [2] H. M. Dintzis (2006) The wandering pathway to determining N to C synthesis of proteins. Some recollections concerning protein structure and biosynthesis, Biochem. Mol. Biol. Educ. 34, 241 246.