Chapter 15: Translation of mrna

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1 Chapter 15: Translation of mrna Student Learning Objectives Upon completion of this chapter you should be able to: 1. Understand the role of translation in gene expression. 2. Understand the genetic basis for protein synthesis and the key experiments which led to the discovery of gene function. 3. Know the features of the genetic code. 4. Know the structure and function of proteins. 5. Understand the role of trna and ribosomes in translation. 6. Understand the initiation, elongation, and termination stages of translation. 7. Distinguish between translation in bacteria and eukaryotes The Genetic Basis for Protein Synthesis The final stage in the information flow from cellular genome to phenotype is the manufacturing of a functional protein. Before going into the details of translation, it is important to get a historical perspective, and this first section does just that. The section considers early experiments which showed that the role of some genes is to encode enzymes. In the early twentieth century, Archibald Garrod, a British physician, studied the inherited disease alkaptonuria (Refer to Figure 15.1). Garrod described the disease as an inborn error of metabolism. Indeed, he was the first to suggest that a connection exists between the function of genes and the production of enzymes. The section then discusses the work of George Beadle and Edward Tatum. In the early 1940s, they studied Neurospora mutants that were altered in their nutritional requirements. A series of experiments such as the one shown in Figure 15.2, allowed them to propose the one-gene/one-enzyme hypothesis. In later decades, this hypothesis had to be modified in three ways: 1) some proteins are not enzymes; 2) some proteins are composed of two or more different polypeptides; and 3) some genes encode RNAs that are not translated into proteins (refer back to Table 14.1). Translation Structural gene Messenger RNA (mrna) Alkaptonuria Inborn error of metabolism One gene-one enzyme hypothesis Protein Polypeptide Focal Points The metabolic pathway of phenylalanine breakdown (Figure 15.1) An example of an experiment that supported Beadle and Tatum s one-gene/one-enzyme hypothesis (Figure 15.2) 169

2 Exercises and Problems Complete the following sentences with the most appropriate word or phrase: (1) was the first researcher to suggest that a relationship exists between the function of a gene and the production of an enzyme. He studied the disease (2), which he described as an inborn error of metabolism. In this disorder, the patient s body accumulates abnormal levels of (3) acid, which is excreted in urine. The one-gene one-enzyme hypothesis was introduced in the 1940s by (4). They studied the inheritance of defects in the metabolic pathways of (5), The term (6) refers to a structure; it is a linear sequence of amino acids. By comparison, the term (7) denotes function The Relationship Between the Genetic Code and Protein Synthesis This section begins by exploring the relationship between transcription and protein synthesis (Figure 15.3). You should use this figure as a reference for the remainder of the chapter. In order for the genetic information, which is written as nucleic acids, to be converted to a functional protein, a code must be present so that groups of nucleotides dictate specific amino acids. This code is presented in Table The section concludes with a discussion of amino acid structure and the linking of amino acids to form three-dimensional proteins. One important concept is the directionality of the mrna to the structure of the polypeptide. Notice that the 5 end of the mrna corresponds to the amino (N-terminal) end of the polypeptide, while the 3 end of the mrna is associated with the C-terminal end (Figure 15.4). You also need to become familiar with the four levels of protein structure (Figure 15.7). This figure is especially important since variations in the genetic information may produce alterations in the protein structure. This may change the phenotype of the organism, alter a metabolic pathway, or in some cases cause lethality. Thus, you need to firmly understand that the DNA and functional proteins are linked. Genetic code Codons Sense codons Start codon Nonsense codons Stop or termination codons Degenerate Synonymous codons Wobble base Reading frame Selenocysteine Pyrrolysine Amino acid Side chain (R group) Peptide bond N-terminus (Amino-terminal end) C-terminus (Carboxyl-terminal end) Primary structure Amino acid sequence Secondary structure -helix -sheet Tertiary structure Quaternary structure Subunits Chaperones 170

3 Focal Points The genetic code (Table 15.1) The relationship between DNA, mrna, trna, and polypeptide (Figure 15.3) The directionality of polypeptide synthesis (Figure 15.4) Levels of structures formed in proteins (Figure 15.7) Exercises and Problems For questions 1 to 6, match each of the following terms that are associated with the genetic code to its correct definition. You may need to refer to Table 15.1 in the text for information. 1. Universal 2. Codons 3. Start codon 4. Stop codon 5. Degenerate 6. Wobble base a. There are very few exceptions to the genetic code in living organisms. b. A group of three nucleotides that specify a single amino acid. c. The codon AUG. d. The fact that the codons UAU and UAC code for the same amino acid. e. The codon UAA. f. There are more codons in the genetic code than there are amino acids. For questions 7 to 12, select the level of protein structure that each statement refers to. a. amino acid structure b. primary structure c. secondary structure d. tertiary structure e. quaternary structure 7. α-helices and β-sheets 8. The linear sequence of amino acids in a polypeptide 9. R groups 10. Hydrophobic and ionic interactions 11. A carboxyl and amino functional group 12. The interaction of two or more polypeptides For questions 13 to 15, complete the sentence with the most appropriate term(s): 13. The 21 st and 22 nd amino acids are and. 14. are proteins that bind to polypeptides and facilitate their proper folding. 15. Codons that specify the same amino acids are termed codons. 170

4 15.3 Experimental Determination of the Genetic Code This section takes a close look at the experimental approaches that helped to deduce the genetic code. The discussion begins with Francis Crick s studies of frame-shift mutations in bacteriophage T4 of E. coli. These experiments indicated that the genetic code is read in multiples of three nucleotides (Refer to Table 15.3). The work of Marshall Nirenberg, Severo Ochoa, and H. Gobind Khorana is covered next. They used synthetic RNA and a cell-free translation system to decipher the genetic code (Refer to pages ). The section ends with a discussion of other experiments that helped decipher the genetic code, namely the synthesis of copolymers by Khorana and colleagues (see Table 15.4), and the triple-binding assay of Nirenberg and Linder (see Figure 15.9). Frameshift mutation Cell-free translation system Focal Points Evidence that the genetic code is read in triplets (Table 15.3) Synthetic RNA helped to determine the genetic code (pages ) The triplet-binding assay (Figure 15.9) Exercises and Problems For questions 1 to 4, complete the following sentences with the most appropriate term(s): 1. Francis Crick performed experiments on which indicated that the genetic code is read in triplets. 2. The gain or loss of one or two bases alters the reading frame beyond the point of change, thus causing a mutation. 3. An in vitro translation system is also called a. 4. Synthetic mrnas can be made using the enzyme. For questions 5 to 8, refer to the following information: A researcher mixes together A, G, and C ribonucleotides in a ratio of 3A:2G:1C. A synthetic RNA is then made from this mixture. 5. How many different codons can this mrna contain? 6. Can this mrna contain a start codon? A stop codon? Why or why not? 7. What proportion of the codons is expected to be 5 AGC 3? 8. What proportion of the codons is expected to be 5 GCG 3? 171

5 15.4 Structure and Function of trna The adaptor hypothesis was proposed in the 1950s by Francis Crick. It suggests that trna molecules recognize codons in mrna and carry amino acids to the ribosome. This section of the chapter examines the structure and function of trna molecules. The anticodon of a trna is complementary to a codon in mrna (see Figure 15.10). Transfer RNAs are the translators of the genetic code and so it is important to know their structure (Figure 15.11). Aminoacyl-tRNA synthetases are a group of enzymes that attach the correct amino acid to the trna. This process is called charging of the trna (Figure 15.12). The final part of this section examines the wobble rules, which allow mismatches at the third position in codon-anticodon pairing (Figure 15.13). Adaptor hypothesis Aminoacyl-tRNA synthetases Aminoacyl trna Charged trna Anticodon Wobble rules Isoacceptor trnas Focal Points Recognition between trnas and mrna (Figure 15.10) Secondary structure of trna (Figure 15.11) Catalytic function of aminoacyl-trna synthetase (Figure 15.12) Wobble position and base-pairing rules (Figure 15.13) Exercises and Problems For questions 1 to 5, match the definition to its correct term. 1. The enzyme that attaches the amino acids to the trna. 2. According to the AT/GC rule, the complementary sequence to the codon. 3. The idea that trnas recognize specific regions of the mrna. 4. The third base of the anticodon can tolerate mismatches. 5. Two or more trnas that are recognized by the same codon. a. aminoacyl-trna synthetase b. adaptor hypothesis c. wobble hypothesis d. anticodon e. isoacceptor trnas 172

6 For questions 6 to 11, refer to the labels in the figure below. 6. The label that indicates the location of the anticodon. 7. The label that indicates the acceptor stem. 8. The 5 end of the trna. 9. The site that will directly interact with the mrna. 10. The 3 end of the trna. 11. A charged trna will have an amino acid attached at this point Ribosome Structure and Assembly Before proceeding to the stages of translation it is first necessary to study the ribosome, which is the site of translation. A ribosome consists of a large and small subunit, each of which is a composite of protein and ribosomal RNA (rrna). The purpose of the ribosome is to provide a workspace for the interaction of the mrna and trna, and to ensure that the mrna is being read in the correct sequence. Nucleolus Ribosome Ribosomal RNA (rrna) Aminoacyl (A) site Peptidyl (P) site Exit (E) site 172

7 Focal Points Composition of bacterial and eukaryotic ribosomes (Table 15.5) Ribosomal structure (Figure 15.14) Exercises and Problems For questions 1 to 5, match the term with its correct definition. 1. Ribosome a. A site where the ribosomal subunits are assembled. 2. P site b. A site on the ribosome where uncharged trnas bind. 3. E site c. The site of translation. 4. A site d. The aminoacyl site. 5. Nucleolus e. The peptidyl site Stages of Translation After introducing the genetic code, trnas and the ribosome, we are prepared to discuss the process of translation. Note that translation involves three stages initiation, elongation, and termination. Although this is the same terminology as was used in transcription, the processes are very different. Before entering into the details of the translation process, take a moment to review Figure Notice the interaction of the mrna, ribosome, and trnas. Once you are familiar with the overview, you can proceed into the more detailed examinations of protein synthesis. During the initiation stage of bacterial translation, the mrna and initiator trna associate with the ribosomal subunits. The Shine-Dalgarno sequence promotes the binding of the mrna to the small ribosomal subunit (Figure 15.17). Elongation involves a repetitive cycle during which successive amino acids are brought into the ribosome (via the A site) and linked together with peptide bonds by peptidyl transferase (Figure 15.18). The growing polypeptide chain is typically located in the P site, and uncharged trnas leave the ribosome through the E site. During the termination stage, the stop codon in mrna is reached and polypeptide synthesis ends. Note that all stages of translation require protein factors, and energy in the form of GTP. Initiation Initiator trna Shine-Dalgarno sequence Kozak s rules Elongation Decoding function Peptidyl transfer Peptidyltransferase Termination Release factors Coupling Polyribosome Polysome 174

8 Focal Points of translation (Figure 15.15) The initiation (Figure 15.16), elongation (Figure 15.18), and termination (Figure 15.19) stages of bacterial translation Translational protein factors in bacteria and eukaryotes (Table 15.6) Coupling between transcription and translation in bacteria (Figure 15.20) Comparison of bacterial and eukaryotic translation (Table 15.7) Exercises and Problems For questions 1 to 7, indicate whether the statement is associated with prokaryotic (P) or eukaryotic (E) translation, or both (B). 1. Initiation factors are required. 2. A 7-methylguanosine cap on the mrna enhances initiation. 3. The mrna binds to the ribosomal subunit at the Shine-Dalgarno sequence. 4. Sorting of the proteins occurs either during or after translation. 5. Termination involves a single release factor. 6. Transcription and translation occur simultaneously. 7. Polyribosomes may occur on the mrna. For each of the following, indicate whether the statement is associated with initiation (I), elongation (E), or termination (T) of translation. 8. IF proteins stabilize the mrna and ribosomal subunits. 9. Nonsense codons enter into the A site. 10. Release factors interact with stop codons. 11. The ribosomal subunits associate with the mrna. 12. The ribosomal subunits dissociate. 13. The peptidyl transfer reaction occurs between the amino acids of the P and A sites. 14. The decoding function of translation occurs. 15. Translocation of the ribosome occurs. For each of the following, indicate whether the statement applies to the aminoacyl (A), exit (E), or peptidyl (P) site of the ribosome. 16. The initiator trna enters through this site. 17. All other charged trnas enter through this site. 18. Uncharged trnas leave through this site. 19. Translation ends when a stop codon appears in this site. 20. The site that is located in the middle of the ribosome. 174

9 Chapter Quiz 1. Hydrophobic and ionic interactions between the R groups of amino acids occur at which level of protein structure? a. primary b. secondary c. tertiary d. quaternary 2. The anticodon is located on the. a. ribosome b. mrna c. trna d. rrna 3. The molecule that attaches the amino acid to the trna is called. a. peptidyltransferase b. release factor c. aminoacyl-trna transferase d. aminoacyl-trna synthetase 4. In which of the following organisms can simultaneous transcription and translation occur? a. prokaryotes b. eukaryotes c. both a and b d. neither a nor b 5. Translocation of the ribosome occurs during. a. elongation b. termination c. initiation d. transcription 6. The Shine-Dalgarno sequence is involved in what stage of translation? a. Initiation in bacteria b. Elongation in bacteria c. Initiation in eukaryotes d. Termination in eukaryotes e. none of the above 7. Which of the following is NOT a stop codon in most species? a. UAA b. UAC c. UGA d. UAG 8. Which of the following statements regarding the genetic code is FALSE? a. It is nearly universal. b. It is degenerate. c. It allows wobble in the third base. d. All of the above are correct. 175

10 9. If the genetic code was a quadruplet (not triplet) code, how many codons would you expect? a. 64 b. 128 c. 256 d During the peptidyl transferase reaction, the ribosomal receives the polypeptide chain. a. A site b. P site c. E site d. S site Answer Key for Study Guide Questions This answer key provides the answers to the exercises and chapter quiz for this chapter. Answers in parentheses ( ) represent possible alternate answers to a problem, while answers marked with an asterisk (*) indicate that the response to the question may vary Garrod 2. alkaptonuria 3. homogentisic 4. Beadle and Tatum 5. Neurospora crassa 6. polypeptide 7. protein a 2. b 3. c 4. e 5. f 6. d 7. c 8. b 9. a 10. d 11. a 12. e 13. selenocysteine and pyrrolysine 14. chaperones 15. synonymous bacteriophages T4 2. frameshift 3. cell-free translation system 4. polynucleotide phosphorylase = No and No; because there are no U s in the mixture 7. (3/6)(2/6)(1/6) = 6/216 or 2.78% 8. (2/6)(1/6)(2/6) = 4/216 or 1.85% 176

11 a 2. d 3. b 4. c 5. e 6. d 7. c 8. b 9. d 10. a 11. a c 2. e 3. b 4. d 5. a B 2. E 3. P 4. E 5. E 6. P 7. B 8. I 9. T 10. T 11. I 12. T 13. E 14. E 15. E 16. P 17. A 18. E 19. A 20. P Quiz 1. c 2. c 3. d 4. a 5. a 6. a 7. b 8. d 9. c 10. a 272