Chapter 27 Protein Metabolism Multiple Choice Questions The genetic code Page: 1038 Difficulty: Ans: C A certain bacterial mRNA is known to represent only one gene and to contain about 800 nucleotides If you assume that the average amino acid residue contributes 110 to the peptide molecular weight, the largest polypeptide that this mRNA could code for would have a molecular weight of about: A) B) C) D) E) 800 5,000 30,000 80,000 An upper limit cannot be determined from the data given The genetic code Page: 1038 Difficulty: Ans: C Assuming that the average amino acid residue contributes 110 to the peptide molecular weight, what will be the minimum length of the mRNA encoding a protein of molecular weight 50,000? A) B) C) D) E) 133 nucleotides 460 nucleotides 1,400 nucleotides 5,000 nucleotides A minimum length cannot be determined from the data given The genetic code Pages: 1039-1044 Difficulty: Ans: D Which of the following are features of the wobble hypothesis? A) B) C) D) E) A naturally occurring tRNA exists in yeast that can read both arginine and lysine codons A tRNA can recognize only one codon Some tRNAs can recognize codons that specify two different amino acids, if both are nonpolar The “wobble” occurs only in the first base of the anticodon The third base in a codon always forms a normal Watson-Crick base pair The genetic code Page: 1039 Difficulty: Ans: C Which one of the following is true about the genetic code? A) B) C) D) All codons recognized by a given tRNA encode different amino acids It is absolutely identical in all living things Several different codons may encode the same amino acid The base in the middle position of the tRNA anticodon sometimes permits “wobble” base pairing with or different codons E) The first position of the tRNA anticodon is always adenosine 314 Chapter 27 Protein Metabolism Protein synthesis Page: 1045 Difficulty: Ans: D Which one of the following statements about ribosomes is true? A) B) C) D) E) The large subunit contains rRNA molecules, the small subunit does not The RNA in ribosomes plays a structural, not catalytic, role There are about 25 of them in an E coli cell There are two major subunits, each with multiple proteins They are relatively small, with molecular weights less than 10,000 Protein synthesis Page: 1049 Difficulty: Ans: A Which of the following statements about tRNA molecules is false? A) A, C, G, and U are the only bases present in the molecule B) Although composed of a single strand of RNA, each molecule contains several short, doublehelical regions C) Any given tRNA will accept only one specific amino acid D) The amino acid attachment is always to an A nucleotide at the 3' end of the molecule E) There is at least one tRNA for each of the 20 amino acids Protein synthesis Page: 1050 Difficulty: Ans: E Which of the following statements about the tRNA that normally accepts phenylalanine is false? (mRNA codons for phenylalanine are UUU and UUC.) A) B) C) D) E) It interacts specificially with the Phe synthetase It will accept only the amino acid phenylalanine Its molecular weight is about 25,000 Phenylalanine can be specifically attached to an —OH group at the 3' end The tRNA must contain the sequence UUU Protein synthesis Page: 1051 Difficulty: Ans: E Which of the following is not true of tRNA molecules? A) B) C) D) E) The 3'-terminal sequence is —CCA Their anticodons are complementary to the triplet codon in the mRNA They contain more than four different bases They contain several short regions of double helix With the right enzyme, any given tRNA molecule will accept any of the 20 amino acids Protein synthesis Page: 1051 Difficulty: Ans: A Aminoacyl-tRNA synthetases (amino acid activating enzymes): A) B) C) D) E) “recognize” specific tRNA molecules and specific amino acids in conjunction with another enzyme attach the amino acid to the tRNA interact directly with free ribosomes occur in multiple forms for each amino acid require GTP to activate the amino acid Chapter 27 Protein Metabolism 315 10 Protein synthesis Page: 1051 Difficulty: Ans: B In E coli, aminoacyl-tRNA synthetases: A) B) C) D) E) activate amino acids in 12 steps are amino acid–specific; there is at least one enzyme specific for each amino acid fall into two classes, each of which attaches amino acids to different ends of the tRNA have no proofreading activities require a tRNA, an amino acid, and GTP as substrates 11 Protein synthesis Pages: 1051-1054 Difficulty: Ans: D Which of the following statements about aminoacyl-tRNA synthetases is false? A) B) C) D) E) Some of the enzymes have an editing/proofreading capability The enzyme attaches an amino acid to the 3' end of a tRNA The enzyme splits ATP to AMP + PPi The enzyme will use any tRNA species, but is highly specific for a given amino acid There is a different synthetase for every amino acid 12 Protein synthesis Page: 1051 Difficulty: Ans: D The enzyme that attaches an amino acid to a tRNA (aminoacyl-tRNA synthetase): A) B) C) D) E) always recognizes only one specific tRNA attaches a specific amino acid to any available tRNA species attaches the amino acid at the 5' end of the tRNA catalyzes formation of an ester bond splits ATP to ADP + Pi 13 Protein synthesis Page: 1051 Difficulty: Ans: D In the “activation” of an amino acid for protein synthesis: A) B) C) D) E) leucine can be attached to tRNAPhe, by the aminoacyl-tRNA synthetase specific for leucine methionine is first formylated, then attached to a specific tRNA the amino acid is attached to the 5' end of the tRNA through a phosphodiester bond there is at least one specific activating enzyme and one specific tRNA for each amino acid two separate enzymes are required, one to form the aminoacyl adenylate, the other to attach the amino acid to the tRNA 14 Protein synthesis Page: 1056 Difficulty: Ans: A Formation of the ribosomal initiation complex for bacterial protein synthesis does not require: A) B) C) D) E) EF-Tu formylmethionyl tRNAfMet GTP initiation factor (IF-2) mRNA 316 Chapter 27 Protein Metabolism 15 Protein synthesis Page: 1058 Difficulty: Ans: D In bacteria the elongation stage of protein synthesis does not involve: A) B) C) D) E) aminoacyl-tRNAs EF-Tu GTP IF-2 peptidyl transferase 16 Protein synthesis Page: 1058 Difficulty: Ans: E Which one of the following statements about the elongation phase of protein synthesis is true? A) B) C) D) At least five high-energy phosphoryl groups are expended for each peptide bond formed During elongation, incoming aminoacylated tRNAs are first bound in the P site Elongation factor EF-Tu facilitates translocation Peptidyl transferase catalyzes the attack of the carboxyl group of the incoming amino acid on an ester linkage in the nascent polypeptide E) Peptidyl transferase is a ribozyme 17 Protein synthesis Page: 1059 Difficulty: Ans: C Which of the following statements about bacterial mRNA is true? A) B) C) D) E) A ribosome usually initiates translation near the end of the mRNA that is synthesized last An mRNA is never degraded but is passed on to the daughter cells at cell division During polypeptide synthesis, ribosomes move along the mRNA in the direction 5' → 3' Ribosomes cannot initiate internally in a polycistronic transcript The codon signaling peptide termination is located in the mRNA near its 5' end 18 Protein synthesis Page: 1059 Difficulty: Bacterial ribosomes: A) B) C) D) E) Ans: B bind tightly to specific regions of DNA, forming polysomes contain at least one catalytic RNA molecule (ribozyme) contain three species of RNA and five different proteins have specific, different binding sites for each of the 20 tRNAs require puromycin for normal function 19 Protein synthesis Page: 1062 Difficulty: Ans: B The large structure consisting of a mRNA molecule being translated by multiple copies of the macromolecular complexes that carry out protein synthesis is called a: A) B) C) D) E) lysosome polysome proteosome ribosome synthosome Chapter 27 Protein Metabolism 317 20 Protein synthesis Page: 1061 Difficulty: Ans: C It is possible to convert the Cys that is a part of Cys-tRNACys to Ala by a catalytic reduction If the resulting Ala-tRNACys were added to a mixture of (1) ribosomes, (2) all the other tRNAs and amino acids, (3) all of the cofactors and enzymes needed to make protein in vitro, and (4) mRNA for hemoglobin, where in the newly synthesized hemoglobin would the Ala from Ala-tRNACys be incorporated? A) B) C) D) E) Nowhere; this is the equivalent of a nonsense mutation Wherever Ala normally occurs Wherever Cys normally occurs Wherever either Ala or Cys normally occurs Wherever the dipeptide Ala-Cys normally occurs 21 Protein synthesis Pages: 1066-1067 Difficulty: Ans: C Which one of the following antibiotics does not function by interfering with the translational process? A) B) C) D) E) Chloramphenicol Cycloheximide Penicillin Puromycin Streptomycin 22 Protein targeting and degradation Page: 1069 Difficulty: Ans: D Which of the following is true about the sorting pathway for proteins destined for incorporation into lysosomes or the plasma membrane of eukaryotic cells? A) B) C) D) Binding of SRP to the signal peptide and the ribosome temporarily accelerates protein synthesis The newly synthesized polypeptides include a signal peptide at their carboxyl termini The signal peptide is cleaved off inside the mitochondria by signal peptidase The signal recognition particle (SRP) binds to the signal peptide soon after it appears outside the ribosome E) The signal sequence is added to the polypeptide in a posttranslational modification reaction 23 Protein targeting and degradation Pages: 1069-1070 Difficulty: Ans: A Glycosylation of proteins inside the endoplasmic reticulum does not involve: A) B) C) D) E) a His residue on the protein an Asn residue on the protein dolichol phosphate glucose N-acetylglucosamine 318 Chapter 27 Protein Metabolism 24 Protein targeting and degradation Page: 1070 Difficulty: Ans: E Posttranslational glycosylation of proteins is inhibited specifically by: A) B) C) D) E) chloramphenicol cycloheximide puromycin streptomycin tunicamycin 25 Protein targeting and degradation Page: 1071 Difficulty: Ans: D The signal sequences that direct proteins to the nucleus are: A) B) C) D) E) always at the amino terminus of the targeted protein cleaved after the protein arrives in the nucleus glycosyl moieties containing mannose 6-phosphate residues not located at the ends of the peptide, but in its interior the same as those that direct certain proteins to lysosomes 26 Protein targeting and degradation Pages: 1072-1074 Difficulty: Ans: C The pathway for polypeptides exported from E coli includes the following steps, which occur in what order for correct export? A) B) C) D) E) A chaperone, SecA, binds to the polypeptide A chaperone, SecB, binds to the polypeptide ATP is hydrolyzed by Sec A SecA pushes 20 amino acids of the polypeptide into the translocation complex 1, 2, 3, 1, 2, 4, 2, 1, 4, 2, 3, 1, 3, 1, 4, 27 Protein targeting and degradation Page: 1076 Difficulty: Ans: D Ubiquitin-mediated protein degradation is a complex process, and many of the signals remain unknown One known signal involves recognition of amino acids in a processed protein that are either stabilizing (Ala, Gly, Met, Ser, etc.) or destabilizing (Arg, Asp, Leu, Lys, Phe, etc.), and are located at: A) B) C) D) E) a helix-turn-helix motif in the protein a lysine-containing target sequence in the protein a zinc finger structure in the protein the amino-terminus of the protein the carboxy-terminus of the protein Chapter 27 Protein Metabolism 319 Short Answer Questions 28 The genetic code Pages: 1035-1036 Difficulty: Outline one of the experimental methods providing evidence that the genetic code was a triplet code Ans: When one or two nucleotides were added to or deleted from a gene, the resulting mRNA produced a protein with a different amino acid sequence after the deletion or insertion When three nucleotides were added or deleted, the resulting protein had a normal sequence except for the insertion or deletion of a single amino acid residue 29 The genetic code Page: 1036 Difficulty: Describe, succinctly, two ways in which synthetic polynucleotides were used in solving the genetic code (you need not describe how the synthetic polynucleotides were made) Ans: (1) When synthetic polymers of only one nucleotide were used as mRNA in vitro, only one of the 20 amino acids was converted into protein For example, poly(U) (containing only the codon UUU) directed the synthesis of polyphenylalanine, showing that UUU encodes Phe (2) Trinucleotides of known sequence were used to stimulate aminoacyl-tRNA binding to ribosomes Because only that aminoacyl-tRNA whose anticodon matched the trinucleotide “mRNA” was bound, the coding specificity of each sequence of three bases could be determined by determining which of the 20 aminoacyl-tRNAs bound (3) Random polymers of RNA containing known ratios of nucleotides (e.g., 70% A and 30% T) generate only certain codons in predictable ratios The identities and ratios of the amino acids specified by such polymers provided important clues that helped solve the genetic code (4) Additional assignments were made possible using synthetic oligonucleotides containing repeats of specific two, three, or four base pair sequences 30 The genetic code Page: 1036 Difficulty: You have isolated a fragment of viral DNA that totally encodes at least two proteins, 120 and 80 amino acids long The DNA fragment is 400 base pairs long (a) Why might you consider this unusual? (b) You sequence the two proteins and find no sequence homology Propose a model to account for these findings Ans: (a) Two distinct proteins of these sizes should require mRNAs of 360 and 240 base pairs because each amino acid residue requires base pairs to code for it (b) No homology means that the smaller protein cannot be derived from the larger by proteolysis; if it were, there would be 80 amino acid residues of identical sequence in the two proteins One possible explanation is that the two genes coding for these proteins overlap and are read in different reading frames 31 The genetic code Page: 1038 Difficulty: The template strand of a segment of double-stranded DNA contains the sequence: (5')CTT TGA TAA GGA TAG CCC TTC (a) What is the base sequence of the mRNA that can be transcribed from this strand? (b) What amino acid sequence could be coded by the mRNA base sequence in (a), using only the first reading frame starting at the 5' end? (Refer to Fig 27-7, p 1038.) (c) Suppose the other (complementary) strand is used as a template for transcription What is the amino acid sequence of the resulting peptide, again starting from the 5' end and using only the first reading frame? 320 Chapter 27 Protein Metabolism Ans: (a) (5')GAA GGG CUA UCC UUA UCA AAG(3’) (b) Glu-Gly-Leu-Ser-Leu-Ser-Lys (c) The codons translate to Leu-Stop-Stop No peptide would be produced because of the stop codons (See also Fig 27-6, p 1038.) 32 The genetic code Page: 1038 Difficulty: Describe the possible outcomes that could occur because of a single base change in an mRNA Ans: The most likely result is a single amino acid change in the encoded protein, when a codon is altered to one of another amino acid However, some nucleotide changes will be “silent” and not change the protein, if the altered codon still specifies the original amino acid Conversion to a nonsense codon will result in a truncated polypeptide, while alteration of the normal stop codon to a “sense” codon will result in a lengthened protein Alternation of the initiaton codon may result in the total failure to translate, and result in no protein product Changes in mRNA sequence outside the protein-coding region may affect translational efficiency, splicing, or mRNA turnover rates 33 The genetic code Page: 1038 Difficulty: The following sequence of four amino acids occurred in the structure of a polypeptide found in a wild-type organism: Leu-Ser-Ile-Arg Several mutants were isolated, each of which carried a single base pair change in the region of DNA that coded for this amino acid sequence Their corresponding amino acid sequences are: Mutant MET-Ser-Ile-Arg Leu-TRP-Ile-Arg Leu-Ser-ARG-Arg Leu-Ser-Ile-PRO Leu-Ser-Ile-TRP What was the nucleotide sequence of the region of mRNA that coded for the amino acid sequence in the wild-type organism? (Refer to Fig 27-6, p 1038.) Ans: (5')C or (5')U UG UCG AUA CGG 34 The genetic code Pages: 1039-1044 Difficulty: In protein synthesis, 61 codons specify the 20 amino acids Base pairing between the codon and the tRNA anticodon assures that the correct amino acid will be inserted into the nascent polypeptide chain Why then does the cell require only 32 different tRNAs to recognize 61 different codons? Ans: Certain tRNAs have the unusual nucleotide inosinate in the first anticodon position Because inosinate can base pair with A, U, or C, a tRNA containing hypoxanthine can recognize three different codons In each recognized codon, there is a standard anticodon-codon base pair with the first two bases of the codon; “wobble” in the third base pair allows one tRNA to read three different codons Similarly, tRNAs with U or G in the first anticodon position also exhibit a wobble effect that permits pairing with two different codons Chapter 27 Protein Metabolism 321 35 Protein synthesis Pages: 1045-1049 Difficulty: Indicate whether the following statements are true (T) or false (F) _A ribosome is the complex within which protein synthesis occurs _Ribosomes contain many separate proteins _The three ribosomal RNAs in a bacterial ribosome are distributed in three separate, large ribosomal subunits _There are four binding sites for aminoacyl-tRNAs on a ribosome Ans: T; T; F; F 36 Protein synthesis Pages: 1051-1053 Difficulty: The process of charging tRNAs with their cognate amino acids involves multiple proofreading steps to increase the overall fidelity Briefly describe these steps Ans: There are two main stages of selection: 1) the synthetase strongly favors activation of the correct amino acid to become aminoacyl-AMP (incorrect amino acids are very poorly activated) and 2) when the correct uncharged tRNA is bound to the enzyme, only the correct aminoacyl-AMP is tolerated in the “proofreading” active site (incorrect aminoacyl-AMPs, though they may become bound, are rapidly hydrolyzed) In addition, for most synthetases, if their tRNA does manage to become acylated by the wrong amino acid, that product is also rapidly hydrolyzed 37 Protein synthesis Pages: 1053-1054 Difficulty: The recognition of an amino acid by its cognate aminoacyl-tRNA synthetase is said to involve a “second genetic code” What is meant by this? Ans: Each of the 20 amino acids has a unique synthetase, but many have multiple cognate tRNAs that must be charged (aminoacylated) The “second code” refers to features common to all tRNAs that carry the same amino acid, making them specifically recognizable by the correct synthetase These features occur at different places in different tRNA classes, and may be as simple as a single G-U basepair, or require ten or more specific nucleotides throughout the sequence (See Fig 27-16, p 1053.) 38 Protein synthesis Page: 1055 Difficulty: In 1961, Howard Dintzis carried out an experiment that defined the direction of polypeptide chain growth during protein synthesis in cells The experiment involved the analysis of hemoglobin molecules that were being synthesized in reticulocytes in the presence of radioactive amino acids Describe the analysis and how it demonstrated the direction of chain growth Ans: In the experiment, only completed polypeptides were isolated and analyzed for the amount of redioactivity in different regions The greatest radioactivity should be located in the region furthest from the initiation point and the least radioactivity in the region closest to the initiation point The opposite is true for the termination point The analysis showed that polypeptide synthesis was initiated at the amino terminus and terminated at the carboxyl terminus 322 Chapter 27 Protein Metabolism 39 Protein synthesis Page: 1056-1058 Difficulty: A given mRNA sequence might be translated in any of three reading frames Describe how prokaryotes and eukaryotes determine the correct reading frame Ans: In prokaryotes, the Shine-Dalgarno sequence in the mRNA base pairs with a complementary sequence in the 16S RNA of the ribosome; this positions the correct start codon (AUG) on the 30S ribosomal subunit Thus, the initiating AUG is distinguished by its proximity to the Shine-Dalgarno sequence In eukaryotes, the initiating AUG codon is the first AUG that the ribosome encounters as it scans the mRNA from its 5' end In both cases the initiating AUG also sets the correct reading frame 40 Protein synthesis Pages: 1056-1061 Difficulty: Match the factor or enzyme at the right with the stage(s) of protein synthesis at which it acts If a factor or enzyme participates in two stages of protein synthesis, indicate both of them _ _ _ _ Amino acid activation Initiation Elongation Termination (a) RF1 (b) EF-Tu (c) aminoacyl-tRNA (d) Shine-Dalgarno sequence Ans: c; d; b; a 41 Protein synthesis Pages: 1056-1061 Difficulty: Indicate whether each of the following statements is true (T) or false (F) _Assembly of a complete ribosome onto an mRNA requires ATP hydrolysis _Aminoacylation or “charging” of tRNA requires the formation of an aminoacyl-AMP intermediate _Aminoacyl-tRNA binding to the A site of the ribosome requires the accessory factor EF-G and GTP hydrolysis _Translocation of a growing polypeptide from the A to the P site on the ribosome requires EFG and GTP hydrolysis _Termination of translation requires release factors, but no NTP hydrolysis Ans: F; T; F; T; T 42 Protein synthesis Pages: 1056-1061 Difficulty: Briefly describe the role of the following components in bacterial protein synthesis (a) Initiation factor (IF-2) (b) 16S RNA (c) Peptidyl transferase (d) Release factors (e) Elongation factor G (EF-G) (f) N10-formyltetrahydrofolate (g) ATP (h) tRNAfMet Chapter 27 Protein Metabolism 323 Ans: (a) IF-2 is a protein factor that, when bound to GTP, brings the fMet-tRNAfMet to the initiation complex (b) 16S RNA is a component of the small (30S) subunit It contains a sequence complementary to the Shine-Dalgarno sequence in the mRNA, and helps to line up the mRNA initiation AUG codon on the ribosome (c) Peptidyl transferase is a ribozyme in the 50S ribosomal subunit It catalyzes formation of each peptide bond as the ribosome moves along the mRNA (d) Release factors are proteins that bring about the release of the finished polypeptide when the ribosome encounters a termination codon in the mRNA (e) EF-G participates in the translocation of the ribosome down the mRNA by one codon after each peptide bond is formed (f) N10-formyltetrahydrofolate is the cofactor that donates a methyl group in the conversion of tRNA-bound Met to fMet (g) ATP is the substrate for aminoacyl-tRNA synthetases; it donates an AMP residue in the formation of aminoacyl adenylate, which donates the aminoacyl group to tRNA (h) tRNAfMet is the transfer RNA that initiates protein synthesis by inserting the first amino acid (fMet) in every prokaryotic protein 43 Protein synthesis Pages: 1056-1061 Difficulty: Number the following steps in the proper order with regard to protein synthesis _ Aminoacyl-tRNA binds to the A site _ Deacylated tRNA is released from ribosome _ Peptide bond formation shifts the growing peptide from the P to the A site _ The 50S subunit binds to the initiation complex of the 30S subunit and mRNA Ans: 2; 4; 3; 44 Protein synthesis Pages: 1056-1062 Difficulty: Indicate whether each of the following statements is true (T) or false (F) _ Bacterial mRNA is broken down within a few minutes of its formation in E coli _ Bacterial mRNA consists only of the bases that code for amino acids _ Polysomes not necessarily contain mRNA _ Bacterial mRNA normally occurs as a double-stranded structure, with one strand containing codons, the other containing anticodons _ Bacterial mRNA can be translated while it is still being synthesized Ans: T; F; F; F; T 45 Protein synthesis Pages: 1057-1058 Difficulty: Ans: B Regarding translation in eukaryotes versus that in prokaryotes (bacteria), indicate whether each of the following statements is true (T) or false (F) _ In eukaryotes the 3' end of the mRNA is associated with the 5' end during initiation whereas in prokaryotes it is not _ In prokaryotes it is initiated at an AUG near a Shine-Dalgarno sequence in the mRNA whereas in eukaryotes it is initiated at an AUG near the 3' end of the mRNA _ In prokaryotes it is initiated with Met whereas in eukaryotes it is initiated with fMet _ In prokaryotes translation and transcription are coupled whereas in eukaryotes they are not Ans: T, F, F, T 324 Chapter 27 Protein Metabolism 46 Protein synthesis Pages: 1058-1061 Difficulty: Polypeptide chain elongation in E coli occurs by the cyclical repetition of three steps What are these steps and what cellular components are necessary for each of them to occur? Ans: The three steps are: (1) An aminoacyl-tRNA is brought to the A site by EF-Tu with bound GTP; (2) peptidyl transferase (a ribozyme) catalyzes peptide-bond formation; (3) the ribosome translocates three nucleotides down the mRNA, helped by EF-G (translocase) This shifts the peptidyl-tRNA to the A site and the deacylated tRNA to the E site 47 Protein synthesis Page: 1060 Difficulty: A new antibiotic was recently discovered that inhibits prokaryotic protein synthesis In the presence of the antibiotic, protein synthesis can be initiated, but only dipeptides that remain bound to the ribosome are formed What specific step of protein synthesis is likely to be blocked by this antibiotic? Ans: The antibiotic probably blocks translocation 48 Protein synthesis Page: 1062 Difficulty: Ans: B The large structure consisting of a mRNA molecule being translated by multiple copies of the macromolecular complexes that carry out protein synthesis is called a: A) B) C) D) E) lysosome polysome proteosome ribosome synthosome 49 Protein synthesis Pages: 1062-1065 Difficulty: Following the synthesis of their polypeptide chain, many proteins require further posttranslational modifications before they attain their full biological activity or function List and describe briefly at least four possible types of modification that can occur Ans: 1) Amino-terminal modification (N-acetylation or deacetylation), 2) removal of signal sequences used for targeting, 3) side-chain modification (phosphorylation, carboxylation, methylation, etc.), 4) attachment of N-linked (to Asn) or O-linked (to Ser or Thr) oligosaccharide moieties, 5) isoprenylation of Cys side-chains, 6) incorporation of prosthetic groups (heme, biotin, etc.), 7) processing of proenzymes or zymogens, and 8) formation of disulfide crosslinks 50 Protein synthesis Page: 1065 Difficulty: In no more than three sentences, describe a nonsense suppressor tRNA and how it differs from a normal tRNA Ans: A suppressor tRNA has one or more altered bases in its anticodon, which allows it to base pair with one of the stop codons This allows the insertion of an amino acid instead of the chain termination that would normally have occurred at the point of a nonsense (stop) codon Chapter 27 Protein Metabolism 325 51 Protein targeting and degradation Page: 1068-1069 Difficulty: When first synthesized, proinsulin has an additional leader or signal peptide at its amino terminus This complete molecule is called preproinsulin and the signal peptide is cleaved off to give proinsulin Briefly, what is the likely function of the signal peptide? Ans: The leader peptide in proinsulin is a signal sequence directing it to the endoplasmic reticulum, from which it enters the Golgi complex and is packaged into a secretory vesicle for secretion by exocytosis 52 Protein targeting and degradation Page: 1069 Difficulty: Describe the sequence of events between the transcription of an mRNA for a secreted protein and the arrival of that protein in the lumen of the endoplasmic reticulum Ans: (1) The mRNA forms an initiation complex with a cytoplasmic ribosome and transcription begins (2) The amino-terminal portion of the nascent chain, containing a signal sequence, binds to an SRP (signal recognition particle), interrupting polypeptide elongation (3) The SRP-ribosomenascent chain complex binds to the ribosome and SRP receptors on the cytosolic face of the ER; the SRP then dissociates (4) Chain elongation continues, with the newly synthesized polypeptide crossing into the ER lumen as it grows (5) The signal sequence is cleaved 53 Protein targeting and degradation Pages: 1071-1073 Difficulty: What are the stages in targeting of nuclear proteins, and why are the targeting sequences not removed upon arrival of the protein in the nucleus? Ans: In the cytoplasm, where eukaryotic protein synthesis occurs, proteins carrying nuclear localization signal (NLS) sequences are bound by a complex of importin α and β, which is then bound to a nuclear pore Ran GTPase mediates translocation of this complex into the nucleus, where importin β dissociates from importin α, and importin α releases the nuclear protein Importin α and β are then exported from the nucleus, and available for another cycle of import The nuclear envelope of higher eukaryotes breaks down at each cell division, distributing the nuclear contents throughout the cell When the nuclear envelope is reestablished, NLS-carrying proteins can be reimported to fulfill their function 54 Protein targeting and degradation Page: 1075 Difficulty: Describe the role of ubiquitin in mediating intracellular protein breakdown Ans: Ubiquitin, a protein found in all eukaryotic cells, is covalently joined to proteins targeted for degradation The carboxyl-terminal residue of ubiquitin is first activated by the formation of a thioester with the first enzyme in the pathway in an ATP-dependent reaction After displacement of the first enzyme by a second, also yielding a thioester link, ubiquitin is finally joined through its carboxyl terminus to a lysine ε-amino group in the protein to be degraded The presence of ubiquitin targets the protein for degradation by cellular proteases