Unit 8 1 UNIT 8 PART A: THE GENETIC CODE PART B: PROTEIN SYNTHESIS PART A: THE GENETIC ODE Genetic experiments in the early 1960's conclusively proved that the genetic code words, or codons, for amino acids are triplets of nucleotides, that the codons do not overlap , and that there is no punctuation betwen codons for succesive amino acid residues. The amino acid sequence of a protein is therefore defined by a linear sequence of contiguous triplet codons. The first AUG codon in a sequence establishes a reading frame, in which a new codon begins at every third nucleotide residue. By 1966 the base sequence of al the triplet code words for each of the amino acids had been deciphered. Asignment: Nelson & Cox, pp. 1065 - 1075. 1. Use a diagram similar to that below to discuss features of the genetic code (pp. 1066 - 1072). .. GAC CAG TCG TG CAC .. .. aa 1 aa 2 aa 3 aa 4 aa 5 .. In doing so, consider: Unit 8 2 a. The coding ratio. Given that there are four diferent bases, what is the minimum number of nucleotides per amino acid required to specify al 20 amino acids (p. 1066)? b. Define "codon" (p. 1066). c. Is the code overlapping or non-overlapping? Use Fig. 27-3 (p. 1066) to distinguish betwen each. d. Use Fig. 27-4 (p. 1067) to define "reading frame". e. Are individual codewords delimited, as for example words in a sentence are delimited by spaces (p. 1067)? f. What establishes the reading frame in RNA (p. 1069)? g. Changes in the nucleotide sequence can profoundly alter the encoded polypeptide. 1) Given a sequence of RNA, an indication of the correct reading frame, and the genetic code (Fig. 27-7, p. 1069), deduce the relevant polypeptide sequence encoded in that reading frame. 2) What are the expected consequences of a one or two base addition or deletion within the above sequence (Fig. 27-4, p. 1067)? 3) Why are such additions or deletions (when they occur in the DNA) caled frameshift mutations? h. Define each of the following (pp. 1069): 1) initiation codon - 2) termination codon - 3) open reading frame i What does degeneracy refer to in the context of coding (Table 27-3, p. 1070)? Unit 8 3 j. Use Fig. 27-8 (p. 1071) to describe how aminoacyl tRNAs interact with mRNA. Point out the anticodon on the tRNA and the codon on the mRNA. k. Is the genetic code almost universal (Box 27-1, pp. 1070 - 1071)? 1) What is the significance of the fact that the amino acid sequence of human insulin is the same, irespective of whether the insulin gene is expresed in humans or in E. coli ? 2) Use Table 1 (Box 27-1, p. 1071) to discuss whether the code is absolutely universal. l. What is meant by "wobble" (p. 1070 - 1072)? m. Use Fig. 27-9 (p. 1073) to explain how retroviruses use translational frameshifting to make more than one polypeptide from a single transcript and to regulate the quantities of each product. n. RNA editing: 1) Use Fig. 27-10 (p. 1073) to show how guide RNAs can be used as templates to direct the addition or removal of specific nucleotides from an mRNA transcript. 2) Use Fig. 27-11 (p. 1073) to show how deaminases can alter specific nucleotides in an mRNA transcript. Use Fig. 27-12 (p. 1074) to show how RNA editing by this mechanism is used to regulate the production of two related but diferent products from a single transcript in a tisue-specific manner. Unit 8 4 PART B: PROTEIN SYNTHESIS Protein synthesis is the most complex of biosynthetic mechanisms and understanding it has been one of the greatest chalenges in the history of biochemistry. Protein synthesis can acount for up to 90% of the chemical energy used by a cel for al biosynthetic reactions. The ribosomes, related protein factors, enzymes, and tRNAs, used in protein synthesis can acount for more than one third of the cel's dry weight. Despite this great complexity, proteins are made at excedingly high rates. A complete polypeptide chain of 100 residues is synthesized in an E. coli cel in about 5 seconds. The synthesis of thousands of diferent proteins in each cel is tightly regulated so that only the required number of molecules of each is made under any given set of metabolic conditions. Asignment: Nelson & Cox, pp. 1075 - 1100. 1. Summarize the key events that occur during each of the following stages of protein synthesis (pp. 1075 - 1076). a. activation of amino acids b. initiation of protein synthesis c. elongation d. termination and ribosome recycling e. folding and post-translational procesing 2. Use Fig. 27-13 (pp. 1077) and Fig. 27-15 (p. 1079) to discuss the molecular composition and thre-dimensional conformation of the ribosome. Unit 8 5 3. Use Fig. 27-14 (p. 1079) to ilustrate that RNA (in addition to proteins) contributes to this thre-dimensional structure of the ribosome. 4. Discuss the ribosome in the context of the RNA world hypothesis (Box 27-2, p. 1078). 5. Why are tRNAs refered to as "adaptors" (p. 1079)? 6. The structure and function of tRNA: Using Fig. 27-17 (p. 1080), point out: a. the anticodon arm (or loop) b. the T?C arm c. the dihydrouracil arm (D arm) d. the 3' (CA) and 5' ends of the tRNA molecule e. the position at which the amino acid is atached f. regions that are hydrogen bonded 7. Amino acid activation (pp. 1081 - 1084) Using structures, describe in two steps how the activation of an amino acid for protein synthesis takes place (Fig. 27-19, p. 1082). Show the structures of the reactants, intermediates, and the products. a. Name the clas of enzyme that loads amino acids onto tRNAs. b. Name the linkage that is broken in ATP (refer to Table 1-15, p. 12)? c. Name the linkage that is formed in the activated intermediate which is then broken in the final stage of the reaction (refer to Table 1-15, p. 12)? d. Name the linkage that is ultimately formed betwen the amino acid and the tRNA (refer to Table 1-15, p. 12)? Unit 8 6 e. What is the other product of this series of reactions? What happens to this product (p. 1081)? f. Write a balanced word equation for the sum of the activation and transfer steps, including hydrolysis of P i (p. 1081) . g. There is a cost of two high energy anhydride bonds in the synthesis of an aminoacyl-tRNA? Acount for this cost (p. 1081). h. Name two other synthetic proceses you have studied in which pyrophosphate is generated. What is the significance of the fact that al cels posses pyrophosphatases that catalyze the hydrolysis reaction, P i + H 2 O ?2P i ? i. List two ends acomplished by aminoacylation of tRNA (p. 1081). j. Discuss how Ile-tRNA synthetase distinguishes betwen Ile and Val at high fidelity (p. 1081 - 1083). k. Use Fig. 27-21 (p. 1083) to discuss what is meant by the "second genetic code". 8. Initiation of translation a. Use Fig. 27-25 (p. 1089) to discuss the key events in translation initiation. b. What is the Shine-Dalgarno sequence (pp. 1088 - 1089)? Use Fig. 27-26 (p. 1090) to explain its significance in prokaryotic translation initiation. c. Acount for the cost of one high energy bond used when IF-2 delivers an aminoacyl tRNA to the P site during translation initiation. d. Discuss the initiation of translation in eukaryotes (Fig. 27-27, p. 1090). Unit 8 7 9. Elongation a. Use Fig. 27-28 (p. 1092) to discuss the role of EF-Tu in delivering A-tRNAs to the A site during elongation. Acount for the one high energy bond used by EF-Tu. What is the role of EF-Ts? b. Use Fig. 27-29 (p. 1092) to discuss peptide bond formation. 1) Name the enzyme and point out the nucleophilic atack mechanism catalyzed by this enzyme. Is this reaction catalyzed by a protein enzyme or a ribozyme? 2) Does protein synthesis run from N to C terminal end -or- C to N? 3) Note the position of the peptidyl tRNA after peptide bond formation. 4) Note: No additional input of energy is required for peptide bond formation because the required energy is released when the peptidyl-tRNA ester bond is broken. c. Use Fig. 27-30 (p. 1093) to discuss translocation. Acount for the one high energy anhydride bond that is used to translocate the peptidyl tRNA from the A site to the P site? d. Discuss the role of EF-Tu in proofreading (pp. 1093 - 1094). 10. Use Fig. 27-31 (p. 1095) to acount for the one high energy bond used in termination. 11. Folding and procesing: Describe some ways that newly synthesized polypeptides are procesed following their synthesis (pp. 1096 - 1098). Unit 8 8 12. Consider a bacterial gene for a hypothetical polypeptide comprised of 100 amino acid residues. a. Approximately how many high energy phosphate anhydride bonds are used to make the necesary mRNA molecule that codes for this protein? b. How many high energy phosphate anhydride bonds are used to synthesize the hypothetical polypeptide molecule from fre amino acids, given the preformed mRNA? c. Asuming that each mRNA molecule is translated 20-50 times, does the cel spend more energy in transcription or translation? 13. Many of the antibiotics used clinicaly are inhibitors of protein synthesis. Discuss why some antibiotics are specificaly used in the treatment of bacterial infections while others are used to treat yeasts and fungi (pp. 1098 - 1099; Note: Mitochondrial and bacterial ribosomes are similar and both are therefore sensitive to bacterial inhibitors.) 14. What is a "signal sequence" and what is its function (p. 1100)? Jim Blankenship Microsoft Word - U08_F08.doc
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