Untitled
CHAPTER 2
Protein Synthesis
9
In Chapter 29 the mechanism of protein synthesis, a process called translation, is ex amined in detail. Translation is a complicated process in which the four-letter alphabet of nucleic acids is translated into the 20-letter alphabet of proteins. The chapter begins with an introduction to the major components of translation—mRNA, tRNA, ribosomes, and aminoacyl-tRNA synthetases. The detailed structures and conformations of tRNAs, the adaptor molecules that recognize both the codons on the mRNAs and the enzymes that attach the corresponding amino acids, are discussed first.Next the authors explain how amino acids are activated for the subsequent formation of peptide bonds through their attachment to tRNAs by the two classes of aminoacyl-tRNA synthetases. The exquisite specificity of these reactions is explored, in terms of correct binding of amino acids and tRNAs to a given synthetase. ThreonyltRNA synthetase is used as an example of specificity at the level of amino acid selection. This enzyme discriminates between threonine and the isosteric valine and the isoelectronic serine, using a combination of selective binding at the active site and proofreading after aminoacylation. Several aminoacyl-tRNA synthetases are used as examples of ways in which the correct tRNA is chosen, ranging from those which require multiple contact points (glutaminyl-tRNA synthetase) to alanyl-tRNA synthetase, which will recognize a “microhelix” containing only the acceptor stem and a hairpin loop.
The authors next turn to the structure and composition of the ribosome, a mo lecular machine that coordinates charged tRNAs, mRNA, and proteins, leading to protein synthesis. The fact that the ribosome is now recognized to be a ribozyme, with the RNA components playing the major role in catalysis, is introduced. The experiments that showed the polarities of polypeptide formation and the translation of mRNA are presented next. Then initiation is described, and the roles of a specialized
517
518
CHAPTER 29 initiator tRNA, the mRNA start codon, and 16S rRNA sequences are outlined. The spatial and functional relationships of the sites on the ribosome that bind aminoacyl-tRNAs and peptidyltRNAs, the peptide-bond–forming reaction, the role of GTP, and the mechanism of the translocation of the peptidyl-tRNA from site to site on the ribosome are presented in the description of the elongation stage of protein synthesis. The wobble hypothesis is then presented to explain the lack of strict one-to-one Watson-Crick base-pairing interactions among the three nucleotides of the tRNA anticodons and the mRNA codons.
The critical role that protein factors play in translation is discussed next, including ini tiation, elongation, and release factors. The termination of translation is outlined, and the role of release factors that recognize translation stop codons is described. The chapter closes with a brief overview of translation in eukaryotes, emphasizing the major contrasting features with respect to translation in prokaryotes. Differences in the initiator tRNA, the selection mechanism of the initiator codon, the ribosomes, and the overall complexity of the process are highlighted. Last, the mechanisms of several potent inhibitors of translation and the mechanism of the bacterial toxin that causes diphtheria is presented.
LEARNING OBJECTIVES
When you have mastered this chapter, you should be able to complete the following objectives.
Introduction 1. Provide an overview of protein synthesis that includes the roles of the amino acids, the tRNAs, the amino acid activating enzymes, mRNA, and the ribosome.
Protein Synthesis Requires the Translation of Nucleotide Sequences
into Amino Acid Sequences (Text Section 29.1) 2. Draw the cloverleaf structure of a tRNA and identify the regions containing the anticodon and the amino acid attachment site.
3. List the features common to all tRNAs.
4. Relate the two-dimensional cloverleaf representation of the tRNA structure to its three dimensional configuration.
Aminoacyl-Transfer RNA Synthetases Read the Genetic Code (Text Section 29.2) 5. Write the two-step reaction sequence of the aminoacyl-tRNA synthetases. Enumerate the high-energy phosphate bonds that are consumed in the overall reaction.
6. Describe the mechanisms of amino acid selection and proofreading that contribute to the accuracy of the attachment of the appropriate amino acid to the correct tRNA.
7. Describe the different modes of recognition of the correct tRNA molecule by aminoacyl tRNA synthetases.
8. Recount an experiment that showed that the tRNA rather than the amino acid in an aminoacyl-tRNA recognizes an mRNA codon.
9. Outline the distinguishing properties of class I and class II aminoacyl-tRNA synthetases.
PROTEIN SYNTHESIS
519
A Ribosome Is a Ribonucleoprotein Particle (70S) Made of a Small (30S)
and a Large (50S) Subunit (Text Section 29.3) 10. List the kinds and numbers of macromolecular components of the prokaryotic ribosome.
Give the mass, sedimentation coefficient, and dimensions of the ribosome of E. coli.
11. Outline the three-dimensional structure of a ribosome.
12. List the evidence that suggests that the RNA components of ribosomes have active roles in protein synthesis.
13. Recount the experiments that established the direction of translation, both in terms of protein synthesis and the reading of mRNA.
14. Name the major initiator codon and the amino acid it encodes. Explain the roles of the nucleotide sequences in 16S rRNA, mRNA, and tRNA in selecting the initiation codon rather than the identical codon that encodes an internal amino acid.
15. Distinguish among the initiator tRNA, tRNAf, and tRNAm and outline the conversion of methionine into formylmethionyl-tRNAf.
16. Explain how some codons are recognized by more than one anticodon, that is, how they in teract with more than one species of aminoacyl-tRNA. List the base-pairing interactions allowed according to the wobble hypothesis.
17. Define the polysome. Correlate the polarity of ribosome movement with the polarity of the growing polypeptide chain.
Protein Factors Play Key Roles in Protein Synthesis (Text Section 29.4) 18. List the components of the 70S initiation complex and indicate the roles of the initiationfactors (IF) and GTP in its formation.
19 Outline the elongation stage of protein synthesis and describe the roles of the elongationfactors (EFs) and GTP in the process. Locate the aminoacyl-tRNAs and peptidyl-tRNAs in the A or P sites of the ribosome during one cycle of elongation.
20. Describe how the GTP–GDP cycle of EF-Tu controls its affinity for its reaction partners.
21. Explain the role of EF-Tu in determining the accuracy and timing of protein synthesis.
22. Outline the translocation steps that occur after the formation of a peptide bond and de scribe the roles of EF-G and GTP.
23. Name the translation stop codons, describe the termination of translation, and explain the roles of the release factors (RFs) in the process.
Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis
Primarily in Translation Initiation (Text Section 29.5) 24. Contrast eukaryotic and prokaryotic ribosomes with respect to composition and size.
25. Contrast the mechanisms of translation initiation in prokaryotes and eukaryotes. Note the different initiator tRNAs, AUG codon selection mechanisms, and numbers of IFs and RFs.
26. Describe the mechanism by which the diphtheria toxin inhibits protein synthesis in eukaryotes.
27. Provide examples of antibiotics that inhibit translation, and describe their mechanisms of action.
520
CHAPTER 29
SELF-TEST
Protein Synthesis Requires the Translation of Nucleotide Sequences
into Amino Acid Sequences 1. Which of the following statements about functional tRNAs are correct?
(a) They contain many modified nucleosides.
(b) About half their nucleosides are in base-paired helical regions.
(c) They contain fewer than 100 ribonucleosides.
(d) Their anticodons and amino acid accepting regions are within 5 Å of each other.
(e) They consist of two helical stems that are joined by loops to form a U-shaped structure.
(f) They have a terminal AAC sequence at their amino acid accepting end.
2. Explain why tRNA molecules must have both unique and common structural features.
Aminoacyl-Transfer RNA Synthetases Read the Genetic Code 3. Which of the following statements about the aminoacyl-tRNA synthetase reaction are correct?
(a) ATP is a cofactor.
(b) GTP is a cofactor.
(c) The amino acid is attached to the 2′- or 3′-hydroxyl of the nucleotide cofactor (ATP).
(d) The amino group of the amino acid is activated.
(e) A mixed anhydride bond is formed.
(f) An acyl ester bond is formed.
(g) An acyl thioester bond is formed.
(h) A phosphoamide (P–N) bond is formed.
4. The DG°′ of the reaction catalyzed by the aminoacyl-tRNA synthetases is (a) ~0 kcal/mol.
(b)
Protein Synthesis
9
In Chapter 29 the mechanism of protein synthesis, a process called translation, is ex amined in detail. Translation is a complicated process in which the four-letter alphabet of nucleic acids is translated into the 20-letter alphabet of proteins. The chapter begins with an introduction to the major components of translation—mRNA, tRNA, ribosomes, and aminoacyl-tRNA synthetases. The detailed structures and conformations of tRNAs, the adaptor molecules that recognize both the codons on the mRNAs and the enzymes that attach the corresponding amino acids, are discussed first.Next the authors explain how amino acids are activated for the subsequent formation of peptide bonds through their attachment to tRNAs by the two classes of aminoacyl-tRNA synthetases. The exquisite specificity of these reactions is explored, in terms of correct binding of amino acids and tRNAs to a given synthetase. ThreonyltRNA synthetase is used as an example of specificity at the level of amino acid selection. This enzyme discriminates between threonine and the isosteric valine and the isoelectronic serine, using a combination of selective binding at the active site and proofreading after aminoacylation. Several aminoacyl-tRNA synthetases are used as examples of ways in which the correct tRNA is chosen, ranging from those which require multiple contact points (glutaminyl-tRNA synthetase) to alanyl-tRNA synthetase, which will recognize a “microhelix” containing only the acceptor stem and a hairpin loop.
The authors next turn to the structure and composition of the ribosome, a mo lecular machine that coordinates charged tRNAs, mRNA, and proteins, leading to protein synthesis. The fact that the ribosome is now recognized to be a ribozyme, with the RNA components playing the major role in catalysis, is introduced. The experiments that showed the polarities of polypeptide formation and the translation of mRNA are presented next. Then initiation is described, and the roles of a specialized
517
518
CHAPTER 29 initiator tRNA, the mRNA start codon, and 16S rRNA sequences are outlined. The spatial and functional relationships of the sites on the ribosome that bind aminoacyl-tRNAs and peptidyltRNAs, the peptide-bond–forming reaction, the role of GTP, and the mechanism of the translocation of the peptidyl-tRNA from site to site on the ribosome are presented in the description of the elongation stage of protein synthesis. The wobble hypothesis is then presented to explain the lack of strict one-to-one Watson-Crick base-pairing interactions among the three nucleotides of the tRNA anticodons and the mRNA codons.
The critical role that protein factors play in translation is discussed next, including ini tiation, elongation, and release factors. The termination of translation is outlined, and the role of release factors that recognize translation stop codons is described. The chapter closes with a brief overview of translation in eukaryotes, emphasizing the major contrasting features with respect to translation in prokaryotes. Differences in the initiator tRNA, the selection mechanism of the initiator codon, the ribosomes, and the overall complexity of the process are highlighted. Last, the mechanisms of several potent inhibitors of translation and the mechanism of the bacterial toxin that causes diphtheria is presented.
LEARNING OBJECTIVES
When you have mastered this chapter, you should be able to complete the following objectives.
Introduction 1. Provide an overview of protein synthesis that includes the roles of the amino acids, the tRNAs, the amino acid activating enzymes, mRNA, and the ribosome.
Protein Synthesis Requires the Translation of Nucleotide Sequences
into Amino Acid Sequences (Text Section 29.1) 2. Draw the cloverleaf structure of a tRNA and identify the regions containing the anticodon and the amino acid attachment site.
3. List the features common to all tRNAs.
4. Relate the two-dimensional cloverleaf representation of the tRNA structure to its three dimensional configuration.
Aminoacyl-Transfer RNA Synthetases Read the Genetic Code (Text Section 29.2) 5. Write the two-step reaction sequence of the aminoacyl-tRNA synthetases. Enumerate the high-energy phosphate bonds that are consumed in the overall reaction.
6. Describe the mechanisms of amino acid selection and proofreading that contribute to the accuracy of the attachment of the appropriate amino acid to the correct tRNA.
7. Describe the different modes of recognition of the correct tRNA molecule by aminoacyl tRNA synthetases.
8. Recount an experiment that showed that the tRNA rather than the amino acid in an aminoacyl-tRNA recognizes an mRNA codon.
9. Outline the distinguishing properties of class I and class II aminoacyl-tRNA synthetases.
PROTEIN SYNTHESIS
519
A Ribosome Is a Ribonucleoprotein Particle (70S) Made of a Small (30S)
and a Large (50S) Subunit (Text Section 29.3) 10. List the kinds and numbers of macromolecular components of the prokaryotic ribosome.
Give the mass, sedimentation coefficient, and dimensions of the ribosome of E. coli.
11. Outline the three-dimensional structure of a ribosome.
12. List the evidence that suggests that the RNA components of ribosomes have active roles in protein synthesis.
13. Recount the experiments that established the direction of translation, both in terms of protein synthesis and the reading of mRNA.
14. Name the major initiator codon and the amino acid it encodes. Explain the roles of the nucleotide sequences in 16S rRNA, mRNA, and tRNA in selecting the initiation codon rather than the identical codon that encodes an internal amino acid.
15. Distinguish among the initiator tRNA, tRNAf, and tRNAm and outline the conversion of methionine into formylmethionyl-tRNAf.
16. Explain how some codons are recognized by more than one anticodon, that is, how they in teract with more than one species of aminoacyl-tRNA. List the base-pairing interactions allowed according to the wobble hypothesis.
17. Define the polysome. Correlate the polarity of ribosome movement with the polarity of the growing polypeptide chain.
Protein Factors Play Key Roles in Protein Synthesis (Text Section 29.4) 18. List the components of the 70S initiation complex and indicate the roles of the initiationfactors (IF) and GTP in its formation.
19 Outline the elongation stage of protein synthesis and describe the roles of the elongationfactors (EFs) and GTP in the process. Locate the aminoacyl-tRNAs and peptidyl-tRNAs in the A or P sites of the ribosome during one cycle of elongation.
20. Describe how the GTP–GDP cycle of EF-Tu controls its affinity for its reaction partners.
21. Explain the role of EF-Tu in determining the accuracy and timing of protein synthesis.
22. Outline the translocation steps that occur after the formation of a peptide bond and de scribe the roles of EF-G and GTP.
23. Name the translation stop codons, describe the termination of translation, and explain the roles of the release factors (RFs) in the process.
Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis
Primarily in Translation Initiation (Text Section 29.5) 24. Contrast eukaryotic and prokaryotic ribosomes with respect to composition and size.
25. Contrast the mechanisms of translation initiation in prokaryotes and eukaryotes. Note the different initiator tRNAs, AUG codon selection mechanisms, and numbers of IFs and RFs.
26. Describe the mechanism by which the diphtheria toxin inhibits protein synthesis in eukaryotes.
27. Provide examples of antibiotics that inhibit translation, and describe their mechanisms of action.
520
CHAPTER 29
SELF-TEST
Protein Synthesis Requires the Translation of Nucleotide Sequences
into Amino Acid Sequences 1. Which of the following statements about functional tRNAs are correct?
(a) They contain many modified nucleosides.
(b) About half their nucleosides are in base-paired helical regions.
(c) They contain fewer than 100 ribonucleosides.
(d) Their anticodons and amino acid accepting regions are within 5 Å of each other.
(e) They consist of two helical stems that are joined by loops to form a U-shaped structure.
(f) They have a terminal AAC sequence at their amino acid accepting end.
2. Explain why tRNA molecules must have both unique and common structural features.
Aminoacyl-Transfer RNA Synthetases Read the Genetic Code 3. Which of the following statements about the aminoacyl-tRNA synthetase reaction are correct?
(a) ATP is a cofactor.
(b) GTP is a cofactor.
(c) The amino acid is attached to the 2′- or 3′-hydroxyl of the nucleotide cofactor (ATP).
(d) The amino group of the amino acid is activated.
(e) A mixed anhydride bond is formed.
(f) An acyl ester bond is formed.
(g) An acyl thioester bond is formed.
(h) A phosphoamide (P–N) bond is formed.
4. The DG°′ of the reaction catalyzed by the aminoacyl-tRNA synthetases is (a) ~0 kcal/mol.
(b)