Prob lem -based Learning Problem -solving Test: The Mechanism of Protein Synthe s is

- - J o zse f S zebere nyi* Terms to be familiar with before you start to solve the test: protein synthesis, ribosomes, amino acids, peptides, peptide bond, polypeptide chain, N- and Cterminus, hemoglobin, - and -globin chains, radioactive labeling, [3H] and [14C]leucine, cytosol, differential centrifugation, density gradient centrifugation, trypsin, electrophoresis, chromatography 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.

Figure 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).

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 non-radioactive leucine molecules produced by the cell from other compounds. B. Radioactive leucine is not diluted out by non-radioactive 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 [3H]leucine for various periods of time. At the same time, other reticulocyte populations received a prolonged labeling with [14C]leucine. The two cultures of cells were mixed and ribosomal and cytosolic fractions were prepared from them. - and -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 twodimensional separation technique of electrophoresis and chromatography. Figure 2 shows such a peptide map of tryptic digests of radioactively labeled -globin (peptides containing radioactive leucine are only shown). Fingerprint analysis of labeled soluble (cytosolic) -globin molecules were performed after different durations of labeling (4, 7, and 60 minutes), the 3H and 14C radioactivities of the spots shown in Fig. 2 were determined, 3H/14C ratios were calculated for each spot and the peptides were ordered in increasing 3H/14C ratios (see Fig. 3a). A similar analysis was performed on ribosome-bound globin chains (Fig. 3b).

Figure 2. Two-dimensional separation ( fingerprint analysis ) of tryptic digests of rabbit globin chains labeled with radioactive leucine. The fingerprint shows radioactively labeled oligopeptides only (for details see the text).

Figure 3. Distribution of [3H]leucine among tryptic peptides of soluble (a) and ribosome-bound (b) rabbit -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].) 4 ____ What was the aim of the double-labeling? A To identify newly synthesized regions in [3H]leucine pulse-labeled globin chains. B To obtain uniformly labeled [14C] globin chains. C To use 14C-radioactivity for each peptide as a measure of leucine content. D To use 3H/14C ratios for each spot as a measure of relative 3H labeling. E All four statements are true.

The hypothetical mechanisms shown in Fig. 1 would give different kinetics of 3H globin labeling in the experiments described earlier. Let's try to predict these patterns! A B C D E Uniform or nearly uniform labeling of all leucine-containing peptides. Randomly uneven labeling of all leucine-containing peptides. Preferential labeling of C-terminal leucine-containing peptides. Preferential labeling of N-terminal leucine-containing peptides. None of the above patterns can be expected.

5 ____ Mechanism A would give this pattern of labeling of ribosome-bound -chains after brief exposure to [3H]leucine. 6 ____ Mechanism A would give this pattern of labeling of ribosome-bound -chains after long exposure to [3H]leucine. 7 ____ Mechanism A would give this pattern of labeling of cytosolic -chains after brief exposure to [3H]leucine. 8 ____ Mechanism A would give this pattern of labeling of cytosolic -chains after long exposure to [3H]leucine. 9 ____ Mechanism B would give this pattern of labeling of ribosome-bound -chains after brief exposure to [3H]leucine. 10 ____ Mechanism B would give this pattern of labeling of ribosome-bound -chains after long exposure to [3H]leucine. 11 ____ Mechanism B would give this pattern of labeling of cytosolic -chains after brief exposure to [3H]leucine. 12 ____ Mechanism B would give this pattern of labeling of cytosolic -chains after long exposure to [3H]leucine. 13 ____ Mechanism C would give this pattern of labeling of ribosome-bound -chains after brief exposure to [3H]leucine. 14 ____ Mechanism C would give this pattern of labeling of ribosome-bound -chains after long exposure to [3H]leucine. 15 ____ Mechanism C would give this pattern of labeling of cytosolic -chains after brief exposure to [3H]leucine. 16 ____ Mechanism C would give this pattern of labeling of cytosolic -chains after long exposure to [3H]leucine. 17 ____ Mechanism D would give this pattern of labeling of ribosome-bound -chains after brief exposure to [3H]leucine. 18 ____ Mechanism D would give this pattern of labeling of ribosome-bound -chains after long exposure to [3H]leucine. 19 ____ Mechanism D would give this pattern of labeling of cytosolic -chains after brief exposure to [3H]leucine. 20 ____ Mechanism D would give this pattern of labeling of cytosolic -chains after long exposure to [3H]leucine.

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 -globin? D Which labeled peptide has the strongest charge? E Which labeled peptide has the highest molecular weight? To answer this question, -globin uniformly labeled with [14C]leucine was briefly digested with carboxypeptidase, an enzyme that removes amino acids from the C-terminus of proteins. The digested -globin was then mixed with intact, uniformly [3H]leucine-labeled -chains and the mixture was subjected to tryptic fingerprinting. 3H- and 14C-radioactivities were measured in each peptide spot. Table I shows the 3H/14C ratios for all labeled peptides. 22 ____ What conclusion can be drawn from the carboxypeptidase experiment? A The N-terminal amino acid of rabbit -globin is arginine or lysine. B The C-terminal amino acid of rabbit -globin is arginine or lysine. C Peptide 16 gives the C-terminus of rabbit -globin. D Peptide 16 gives the N-terminus of rabbit -globin. E Peptide 16 is closest to the C-terminus among the radioactively labeled tryptic peptides. 23____ We can now describe the exact mechanism of protein synthesis. -globin chains are synthesized. A By simultaneous enzymatic ligation of adjacent amino acids. B By simultaneous enzymatic ligation of preformed short peptides. 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.

ENCES 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.