Chapter Summary, Questions Answers - Protein Synthesis

CHAPTER SUMMARY

Codons are composed of three nucleotide bases presented in the messenger (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U). They are always written 5I →3I . Of the 64 possible three-base combinations, 61 code for the 20 common amino acids and 3 signal termination of protein synthesis (translation). Altering the nucleotide sequence in a codon can cause silent mutations (the altered codon codes for the original amino acid), missense mutations (the altered codon codes for a different amino acid), or nonsense mutations (the altered codon is a termination codon). Characteristics of the genetic code include specificity, universality, and degeneracy, and it is nonoverlapping and commaless (Figure 31.17). Requirements for protein synthesis include all the amino acids that eventually appear in the finished protein, at least one specific type of transfer RNA (tRNA) for each amino acid, one aminoacyl-tRNA synthetase for each amino acid, the mRNA coding for the protein to be synthesized, fully competent ribosomes, protein factors needed for initiation, elongation, and termination of protein synthesis, and ATP and GTP as energy sources. tRNA has an attachment site for a specific amino acid at its 3I -end, and an anticodon region that can recognize the codon specifying the amino acid the tRNA is carrying. Ribosomes are large complexes of protein and ribosomal (rRNA). They consist of two subunits. Each ribosome has three binding sites for tRNA molecules: the A, P, and E sites that cover three neighboring codons. The A-site codon binds an incoming aminoacyl-tRNA, the P-site codon is occupied by peptidyl-tRNA, and the E site is occupied by the empty tRNA as it is about to exit the ribosome. Recognition of an mRNA codon is accomplished by the tRNA anticodon. The anticodon binds to the codon following the rules of complementarity and antiparallel binding. (Nucleotide sequences are always assumed to be written in the 5I to 3I direction unless otherwise noted.) The “wobble” hypothesis states that the first (5I ) base of the anticodon is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the last (3I ) base of the codon, thus allowing a single tRNA to recognize more than one codon for a specific amino acid. For initiation of protein synthesis, the components of the translation system are assembled, and mRNA associates with the small ribosomal subunit. The process requires initiation factors. In prokaryotes, a purine-rich region of the mRNA (the Shine-Dalgarno sequence) base-pairs with a complementary sequence on 16S rRNA, resulting in the positioning of the small subunit on the mRNA so that translation can begin. The 5I -cap (bound by proteins of the eIF-4 family) on eukaryotic mRNA is used to position the small subunit on the mRNA. The initiation codon is AUG, and N-formylmethionine is the initiating amino acid in prokaryotes, whereas methionine is used in eukaryotes. The polypeptide chain is elongated by the addition of amino acids to the carboxyl end of its growing chain. The process requires elongation factors that facilitate the binding of the aminoacyl-tRNA to the A site as well as the movement of the ribosome along the mRNA. The formation of the peptide bond is catalyzed by peptidyltransferase, which is an activity intrinsic to the rRNA of the large subunit and, therefore, is a ribozyme. Following peptide bond formation, the ribosome advances along the mRNA in the 5I →3I direction to the next codon (translocation). Because of the length of most mRNAs, more than one ribosome at a time can translate a message, forming a polysome. Termination begins when one of the three termination codons moves into the A site. These codons are recognized by release factors. The newly synthesized protein is released from the ribosomal complex, and the ribosome is dissociated from the mRNA. Initiation, elongation, and termination are driven by the hydrolysis of GTP. Initiation in eukaryotes also requires ATP for scanning. Numerous antibiotics interfere with the process of protein synthesis. Many polypeptide chains are covalently modified during or after translation. Such modifications include removal of amino acids; phosphorylation, which may activate or inactivate the protein; glycosylation, which plays a role in protein targeting; and hydroxylation such as that seen in collagen. Proteins must fold to achieve their functional form. Folding can be spontaneous or facilitated by chaperones. Proteins that are defective (for example misfolded) or destined for rapid turnover are marked for destruction by the attachment of chains of a small, highly conserved protein called ubiquitin. Ubiquitinated proteins are rapidly degraded by a cytosolic complex known as the proteasome.

Figure 31.17 Key concept map for protein synthesis. mRNA = messenger RNA; tRNA = transfer RNA; A = adenine; G = guanine; C = cytosine; U = uracil.

Study Questions
Choose the ONE best answer.

31.1 A 20-year-old man with a microcytic anemia is found to have an abnormal form of β-globin (Hemoglobin Constant Spring) that is 172 amino acids long, rather than the 141 found in the normal protein. Which of the following point mutations is consistent with this abnormality?

A. CGA → UGA

B. GAU → GAC

C. GCA → GAA

D. UAA → CAA

E. UAA → UAG

Correct answer = D. Mutating the normal termination (stop) codon for β-globin from UAA to CAA causes the ribosome to insert a glutamine at that point. It will continue extending the protein chain until it comes upon the next stop codon further down the message, resulting in an abnormally long protein. The replacement of CGA (arginine) with UGA (stop) would cause the protein to be too short. GAU and GAC both encode aspartate and would cause no change in the protein. Changing GCA (alanine) to GAA (glutamate) would not change the size of the protein product. A change from UAA to UAG would simply change one termination codon for another, and would have no effect on the protein

31.2 A pharmaceutical company is studying a new antibiotic that inhibits bacterial protein synthesis. When this antibiotic is added to an in vitro protein synthesis system that is translating the messenger RNA sequence AUGUUUUUUUAG, the only product formed is the dipeptide fMet-Phe. What step in protein synthesis is most likely inhibited by the antibiotic?

A. Initiation

B. Binding of charged transfer RNA to the ribosomal A site

C. Peptidyltransferase activity

D. Ribosomal translocation

E. Termination

Correct answer = D. Because fMet-Phe is made, the ribosomes must be able to complete initiation, bind Phe-tRNA to the A site, and use peptidyltransferase activity to form the first peptide bond. Because the ribosome is not able to proceed any further, ribosomal movement (translocation) is most likely the inhibited step. The ribosome is, therefore, frozen before it reaches the termination codon of this message.

31.3 A transfer RNA (tRNA) molecule that is supposed to carry cysteine (tRNAcys) is mischarged, so that it actually carries alanine (ala-tRNAcys). Assuming no correction occurs, what will be the fate of this alanine residue during protein synthesis?

A. It will be incorporated into a protein in response to a codon for alanine.

B. It will be incorporated into a protein in response to a codon for cysteine.

C. It will be incorporated randomly at any codon.

D. It will remain attached to the tRNA because it cannot be used for protein synthesis.

E. It will be chemically converted to cysteine by cellular enzymes.

Correct answer = B. Once an amino acid is attached to a transfer (tRNA) molecule, only the anticodon of that tRNA determines the specificity of incorporation. The mischarged alanine will, therefore, be incorporated into the protein at a position determined by a cysteine codon.

31.4 In a patient with cystic fibrosis caused by the ∆F508 mutation, the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein folds incorrectly. The patient s cells modify this abnormal protein by attaching ubiquitin molecules to it. What is the fate of this modified CFTR protein?

A. It performs its normal function because the ubiquitin largely corrects for the effect of the mutation.

B. It is secreted from the cell.

C. It is placed into storage vesicles.

D. It is degraded by the proteasome.

E. It is repaired by cellular enzymes.

Correct answer = D. Ubiquitination usually marks old, damaged, or misfolded proteins for destruction by the cytosolic proteasome. There is no known cellular mechanism for repair of damaged proteins.

31.5 Many antimicrobials inhibit protein translation. Which of the following antimicrobials is correctly paired with its mechanism of action?

A. Erythromycin binds to the 60S ribosomal subunit.

B. Puromycin inactivates EF-2.

C. Streptomycin binds to the 30S ribosomal subunit.

D. Tetracyclines inhibit peptidyltransferase.

Correct answer = C. Streptomycin binds the 30S subunit and inhibits translation initiation. Erythromycin binds the 50S ribosomal subunit (60S denotes a eukaryote) and blocks the tunnel through which the peptide leaves the ribosome. Puromycin has structural similarity to aminoacyl-tRNA. It is incorporated into the growing chain, inhibits elongation, and results in premature termination in both prokaryotes and eukaryotes. Tetracyclines bind the 30S ribosomal subunit and block access to the A site, inhibiting elongation.

31.6 Translation of a synthetic polyribonucleotide containing the repeating sequence CAA in a cell-free protein-synthesizing system produces three homopolypeptides: polyglutamine, polyasparagine, and polythreonine. If the codons for glutamine and asparagine are CAA and AAC, respectively, which of the following triplets is the codon for threonine?

A. AAC

B. ACA

C. CAA

D. CAC

E. CCA

Correct answer = B. The synthetic polynucleotide sequence of CAACAACAACAA.. could be read by the in vitro protein synthesizing system starting at the first C, the first A, or the second A. In the first case, the first triplet codon would be CAA, which codes glutamine; in the second case, the first triplet codon would be AAC, which codes for asparagine; in the last case, the first triplet codon would be ACA, which codes for threonine.

31.7 Which of the following is required for both prokaryotic and eukaryotic protein synthesis?

A. Binding of the small ribosomal subunit to the Shine-Dalgarno sequence

B. fMet-tRNA

C. Movement of the messenger RNA out of the nucleus and into the cytoplasm

D. Recognition of the 5 -cap by initiation factors.

E. Translocation of the peptidyl-tRNA from the A site to the P site

Correct answer = E. In both prokaryotes and eukaryotes, continued translation (elongation) requires movement of the peptidyl-tRNA from the A to the P site to allow the next aminoacyl-tRNA to enter the A site. Only prokaryotes have a Shine-Dalgarno sequence and use fMet, and only eukaryotes have a nucleus and co- and posttranscriptionally process their mRNA.

31. 8 α1-Antitrypsin (AAT) deficiency can result in emphysema, a lung pathology, because the action of elastase, a serine protease, is unopposed. Deficiency of AAT in the lungs is the consequence of impaired secretion from the liver, the site of its synthesis. Proteins such as AAT that are destined to be secreted are best characterized by which of the following statements?

A. Their synthesis is initiated on the smooth endoplasmic reticulum.

B. They contain a mannose 6-phosphate targeting signal.

C. They always contain methionine as the N-terminal amino acid.

D. They are produced from translation products that have an N-terminal hydrophobic signal sequence.

E. They contain no sugars with O-glycosidic linkages because their synthesis does not involve the Golgi apparatus.

Correct answer = D. Synthesis of secreted proteins is begun on free (cytosolic) ribosomes. As the N-terminal signal sequence of the peptide emerges from the ribosome, it is bound by the signal recognition particle, taken to the rough endoplasmic reticulum (RER), threaded into the lumen, and removed as translation continues. The proteins move through the RER and the Golgi, and undergo processing such as N-glycosylation (RER) and O-glycosylation (Golgi). In the Golgi, they are packaged in secretory vesicles and released from the cell. The smooth endoplasmic reticulum is associated with synthesis of lipids, not proteins, and has no ribosomes attached. Phosphorylation at carbon 6 of terminal mannose residues in glycoproteins targets these proteins (acid hydrolases) to lysosomes. The N-terminal methionine is removed from most proteins during processing.

31.9 Why is the genetic code described both as degenerate and unambiguous?

A given amino acid can be coded for by more than one codon (degenerate code), but a given codon codes for just one particular amino acid (unambiguous code).