CHAPTER 7 FROM DNA TO PROTEIN: HOW CELLS READ THE GENOME 2009 Garland Science Publishing From DNA to RNA 7-1 RNA in cells differs from DNA in that ___________________. (a) it contains the base uracil, which pairs with cytosine (b) it is single-stranded and cannot form base pairs (c) it is single-stranded and can fold up into a variety of structures (d) the sugar ribose contains fewer oxygen atoms than does deoxyribose 7-2 Transcription is similar to DNA replication in that ___________________. (a) an RNA transcript is synthesized discontinuously and the pieces are then joined together (b) it uses the same enzyme as that used to synthesize RNA primers during DNA replication (c) the newly synthesized RNA remains paired to the template DNA (d) nucleotide polymerization occurs only in the 5′-to-3′ direction 7-3 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; use each word or phrase only once. For a cell’s genetic material to be used, the information is first copied from the DNA into the nucleotide sequence of RNA in a process called __________________. Various kinds of RNA are produced, each with different functions. __________________ molecules code for proteins, __________________ molecules act as adaptors for protein synthesis, __________________ molecules are integral components of the ribosome, and __________________ molecules are important in the splicing of RNA transcripts. incorporation mRNA pRNA translation 7-4 rRNA snRNA transcription Match the following structures with their names. transmembrane tRNA proteins Figure Q7-4 7-5 Which of the following statements is false? (a) A new RNA molecule can begin to be synthesized from a gene before the previous RNA molecule’s synthesis is completed. (b) If two genes are to be expressed in a cell, these two genes can be transcribed with different efficiencies. (c) RNA polymerase is responsible for both unwinding the DNA helix and catalyzing the formation of the phosphodiester bonds between nucleotides. (d) Unlike DNA, RNA uses a uracil base and a deoxyribose sugar. 7-6 Which one of the following is the main reason that a typical eucaryotic gene is able to respond to a far greater variety of regulatory signals than a typical procaryotic gene or operon? (a) Eucaryotes have three types of RNA polymerase. (b) Eucaryotic RNA polymerases require general transcription factors. (c) The transcription of a eucaryotic gene can be influenced by proteins that bind far from the promoter. (d) Procaryotic genes are packaged into nucleosomes. 7-7 Match the following types of RNA with the main polymerase that transcribes them. 7-8 List three ways in which the process of eucaryotic transcription differs from the process of bacterial transcription. 7-9 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once. In eucaryotic cells, general transcription factors are required for the activity of all promoters transcribed by RNA polymerase II. The assembly of the general transcription factors begins with the binding of the factor __________________ to DNA, causing a marked local distortion in the DNA. This factor binds at the DNA sequence called the __________________ box, which is typically located 25 nucleotides upstream from the transcription start site. Once RNA polymerase II has been brought to the promoter DNA, it must be released to begin making transcripts. This release process is facilitated by the addition of phosphate groups to the tail of RNA polymerase by the factor __________________. It must be remembered that the general transcription factors and RNA polymerase are not sufficient to initiate transcription in the cell and are affected by proteins bound thousands of nucleotides away from the promoter. Proteins that link the distantly bound transcription regulators to RNA polymerase and the general transcription factors include the large complex of proteins called the__________________. The packing of DNA into chromatin also affects transcriptional initiation, and histone __________________ is an enzyme that can render the DNA less accessible to the general transcription factors. activator CAP deacetylase enhancer 7-10 lac ligase mediator TATA TFIIA TFIID TFIIH You have a piece of DNA that includes the following sequence: 5′-ATAGGCATTCGATCCGGATAGCAT-3′ 3′-TATCCGTAAGCTAGGCCTATCGTA-5′ Which of the following RNA molecules could be transcribed from this piece of DNA? (a) (b) (c) (d) 7-11 5′-UAUCCGUAAGCUAGGCCUAUGCUA-3′ 5′-AUAGGCAUUCGAUCCGGAUAGCAU-3′ 5′-UACGAUAGGCCUAGCUUACGGAUA-3′ none of the above You have a segment of DNA that contains the following sequence: 5′-GGACTAGACAATAGGGACCTAGAGATTCCGAAA-3′ 3′-CCTGATCTGTTATCCCTGGATCTCTAAGGCTTT-5′ If you know that the RNA transcribed from this segment contains the following sequence: 5′-GGACUAGACAAUAGGGACCUAGAGAUUCCGAAA–3′ Which of the following choices best describes how transcription occurs? (a) The top strand is the template strand; RNA polymerase moves along this strand from 5′ to 3′. (b) The top strand is the template strand; RNA polymerase moves along this strand from 3′ to 5′. (c) The bottom strand is the template strand; RNA polymerase moves along this strand from 5′ to 3′. (d) The bottom strand is the template strand; RNA polymerase moves along this strand from 3′ to 5′. 7-12 Which of the following molecules of RNA would you predict to be the most likely to fold into a specific structure as a result of intramolecular base pairing? (a) (b) (c) (d) 7-13 5′CCCUAAAAAAAAAAAAAAAAUUUUUUUUUUUUUUUUAGGG-3′ 5′UGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUG-3′ 5′AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3′ 5′GGAAAAGGAGAUGGGCAAGGGGAAAAGGAGAUGGGCAAGG-3′ Imagine that an RNA polymerase is transcribing a segment of DNA that contains the following sequence: 5′-AGTCTAGGCACTGA-3′ 3′-TCAGATCCGTGACT 5′ A. B. 7-14 If the polymerase is transcribing from this segment of DNA from left to right, which strand (top or bottom) is the template? What will be the sequence of that RNA (be sure to label the 5′ and 3′ ends of your RNA molecule)? The sigma subunit of bacterial RNA polymerase ___________________. (a) contains the catalytic activity of the polymerase (b) remains part of the polymerase throughout transcription (c) recognizes promoter sites in the DNA (d) recognizes transcription termination sites in the DNA 7-15 Which of the following might decrease the transcription of only one specific gene in a bacterial cell? (a) a decrease in the amount of sigma factor (b) a decrease in the amount of RNA polymerase (c) a mutation that introduced a stop codon into the DNA that precedes the gene’s coding sequence (d) a mutation that introduced extensive sequence changes into the DNA that precedes the gene’s transcription start site 7-16 There are several reasons why the primase used to make the RNA primer for DNA replication is not suitable for gene transcription. Which of the statements below is not one of those reasons? (a) Primase initiates RNA synthesis on a single-stranded DNA template. (b) Primase can initiate RNA synthesis without the need for a base-paired primer. (c) Primase synthesizes only RNAs of about 5–20 nucleotides in length. (d) The RNA synthesized by primase remains base-paired to the DNA template. 7-17 You have a bacterial strain with a mutation that removes the transcription termination signal from the Abd operon. Which of the following statements describes the most likely effect of this mutation on Abd transcription? (a) The Abd RNA will not be produced in the mutant strain. (b) The Abd RNA from the mutant strain will be longer than normal. (c) Sigma factor will not dissociate from RNA polymerase when the Abd operon is being transcribed in the mutant strain. (d) RNA polymerase will move in a backwards fashion at the Abd operon in the mutant strain. 7-18 Transcription in bacteria differs from transcription in a eucaryotic cell because __________________________. (a) RNA polymerase (along with its sigma subunit) can initiate transcription on its own (b) RNA polymerase (along with its sigma subunit) requires the general transcription factors to assemble at the promoter before polymerase can begin transcription (c) The sigma subunit must associate with the appropriate type of RNA polymerase to produce mRNAs (d) RNA polymerase must be phosphorylated at its C-terminal tail for transcription to proceed 7-19 Which of the following does not occur before a eucaryotic mRNA is exported from the nucleus? (a) The ribosome binds to the mRNA. (b) The mRNA is polyadenylated at its 3′ end. (c) (d) 7-20 7-methyl-G is added in a 5′ to 5′ linkage to the mRNA. RNA polymerase dissociates. Use the numbers in the choices below to indicate where in the schematic diagram of a eucaryotic cell (Figure Q7-20) those processes take place. Figure Q7-20 1. 2. 3. 4. 5. transcription translation RNA splicing polyadenylation RNA capping 7-21 Total nucleic acids are extracted from a culture of yeast cells and are then mixed with resin beads to which the polynucleotide 5′TTTTTTTTTTTTTTTTTTTTTTTTT-3′ has been covalently attached. After a short incubation, the beads are then extracted from the mixture. When you analyze the cellular nucleic acids that have stuck to the beads, which of the following is most abundant? (a) DNA (b) tRNA (c) rRNA (d) mRNA 7-22 Name three covalent modifications that can be made to an RNA molecule in eucaryotic cells before the RNA molecule becomes a mature mRNA. 7-23 Which of the following statements about RNA splicing is false? (a) Conventional introns are not found in bacterial genes. (b) (c) (d) For a gene to function properly, every exon must be removed from the primary transcript in the same fashion on every mRNA molecule produced from the same gene. Small RNA molecules in the nucleus perform the splicing reactions necessary for the removal of introns. Splicing occurs after the 5′ cap has been added to the end of the primary transcript. 7-24 The length of a particular gene in human DNA, measured from the start site for transcription to the end of the protein-coding region, is 10,000 nucleotides, whereas the length of the mRNA produced from this gene is 4000 nucleotides. What is the most likely reason for this difference? 7-25 Why is the old dogma “one gene—one protein” not always true for eucaryotic genes? 7-26 Is this statement true or false? Explain your answer. “Since introns do not contain protein coding information, they do not have to be removed precisely (meaning, a nucleotide here and there should not matter) from the primary transcript during RNA splicing.” 7-27 You have discovered a gene (Figure Q7-27A) that is alternatively spliced to produce several forms of mRNA in various cell types, three of which are shown in Figure Q7-27B. The lines connecting the exons that are included in the mRNA indicate the splicing. From your experiments, you know that protein translation begins in exon 1. For all forms of the mRNA, the encoded protein sequence is the same in the regions of the mRNA that correspond to exons 1 and 10. Exons 2 and 3 are alternative exons used in different mRNA, as are exons 7 and 8. Which of the following statements about exons 2 and 3 is the most accurate? Explain your answer. Figure Q7-27 (a) (b) (c) (d) Exons 2 and 3 must have the same number of nucleotides. Exons 2 and 3 must contain an integral number of codons (that is, the number of nucleotides divided by 3 must be an integer). Exons 2 and 3 must contain a number of nucleotides that when divided by 3, leaves the same remainder (that is, 0, 1, or 2). Exons 2 and 3 must have different numbers of nucleotides. From RNA to Protein 7-28 Which of the following statements about the genetic code is correct? (a) All codons specify more than one amino acid. (b) The genetic code is redundant. (c) All amino acids are specified by more than one codon. (d) All codons specify an amino acid. NOTE: The following codon table is to be used for Problems Q7-29 to Q7-36, Q7-43, and Q7-47. 7-29 The piece of RNA below includes the region that codes the binding site for the initiator tRNA needed in translation. 5′-GUUUCCCGUAUACAUGCGUGCCGGGGGC-3′ Which amino acid will be on the tRNA that is the first to bind to the A-site of the ribosome? (a) methionine (b) arginine (c) cystine (d) valine 7-30 The following DNA sequence includes the beginning of a sequence coding for a protein. What would be the result of a mutation that changed the C marked by an asterisk to an A? 5′-AGGCTATGAATGGACACTGCGAGCCC…. * 7-31 Which amino acid would you expect a tRNA with the anticodon 5′-CUU-3′ to carry? (a) lysine (b) glutamic acid (d) leucine (d) phenylalanine 7-32 Which of the following pairs of codons might you expect to be read by the same tRNA as a result of wobble? (a) CUU and UUU (b) GAU and GAA (c) CAC and CAU (d) AAU and AGU 7-33 Below is a segment of RNA from the middle of an mRNA. 5′-UAGUCUAGGCACUGA-3′ If you were told that this segment of RNA was part of the coding region of an mRNA for a large protein, give the amino acid sequence for the protein that is encoded by this segment of mRNA. Write your answer using the one-letter amino acid code. 7-34 Below is the sequence from the 3′ end of an mRNA. 5′-CCGUUACCAGGCCUCAUUAUUGGUAACGGAAAAAAAAAAAAAA-3′ If you were told that this sequence contains the stop codon for the protein encoded by this mRNA, what is the anticodon on the tRNA in the P-site of the ribosome when release factor binds to the A-site? (a) 5′-CCA-3′ (b) 5′-CCG-3′ (c) 5′-UGG-3′ (d) 5′-UUA-3′ 7-35 One strand of a section of DNA isolated from the bacterium E. coli reads: 5′-GTAGCCTACCCATAGG-3′ A. B. C. 7-36 Suppose that an mRNA is transcribed from this DNA using the complementary strand as a template. What will be the sequence of the mRNA in this region (make sure you label the 5′ and 3′ ends of the mRNA)? How many different peptides could potentially be made from this sequence of RNA, assuming that translation initiates upstream of this sequence? What are these peptides? (Give your answer using the one-letter amino acid code.) A strain of yeast translates mRNA into protein inaccurately. Individual molecules of a particular protein isolated from this yeast have variations in the first 11 amino acids compared with the sequence of the same protein isolated from normal yeast cells, as listed in Figure Q7-36. What is the most likely cause of this variation in protein sequence? Figure Q7-36 (a) (b) (c) (d) a mutation in the DNA coding for the protein a mutation in the anticodon of the isoleucine tRNA (tRNAIle) a mutation in the isoleucyl-tRNA synthetase that decreases its ability to distinguish between different amino acids a mutation in the isoleucyl-tRNA synthetase that decreases its ability to distinguish between different tRNA molecules 7-37 Which of the following statements is true? (a) Ribosomes are large RNA structures composed solely of rRNA. (b) Ribosomes are synthesized entirely in the cytoplasm. (c) rRNA contains the catalytic activity that joins amino acids together. (d) A ribosome binds one tRNA at a time. 7-38 Figure Q7-38A shows the stage in translation when an incoming aminoacyl-tRNA has bound to the A-site on the ribosome. Using the components shown in Figure Q7-38A as a guide, show on Figure Q7-38B and Q7-38C what happens in the next two stages to complete the addition of the new amino acid to the growing polypeptide chain. Figure Q7-38 7-39 A poison added to an in vitro translation mixture containing mRNA molecules with the sequence 5′-AUGAAAAAAAAAAAAUAA-3′ has the following effect: the only product made is a Met-Lys dipeptide that remains attached to the ribosome. What is the most likely way in which the poison acts to inhibit protein synthesis? (a) It inhibits peptidyl transferase activity. (b) It inhibits movement of the small subunit relative to the large subunit. (c) It inhibits release factor. (d) It mimics release factor. 7-40 In eucaryotes, but not in procaryotes, ribosomes find the start site of translation by ____________________________. (a) binding directly to a ribosome-binding site preceding the initiation codon (b) scanning along the mRNA from the 5′ end (c) recognizing an AUG codon as the start of translation (d) binding an initiator tRNA 7-41 Which of the following statements about procaryotic mRNA molecules is false? (a) A single procaryotic mRNA molecule can be translated into several proteins. (b) Ribosomes must bind to the 5′ cap before initiating translation. (c) mRNAs are not polyadenylated. (d) Ribosomes can start translating an mRNA molecule before transcription is complete. 7-42 Figure Q7-42 shows an mRNA molecule. Figure Q7-42 A. B. Match the labels given in the list below with the label lines in Figure Q742. (a) ribosome-binding site (b) initiator codon (c) stop codon (d) untranslated 3′ region (e) untranslated 5′ region (f) protein-coding region Is the mRNA shown procaryotic or eucaryotic? Explain your answer. 7-43 A mutation in the tRNA for the amino acid lysine results in the anticodon sequence 5′-UAU-3′ (instead of 5′-UUU-3′). Which of the following aberrations in protein synthesis might this tRNA cause? (Refer to the codon table provided above Q7-29.) (a) read-through of stop codons (b) substitution of lysine for isoleucine (c) substitution of lysine for tyrosine (d) substitution of lysine for phenylalanine 7-44 You have discovered a protein that inhibits translation. When you add this inhibitor to a mixture capable of translating human mRNA and centrifuge the mixture to separate polyribosomes and single ribosomes, you obtain the results shown in Figure Q7-44. Which of the following interpretations is consistent with these observations? Figure Q7-44 (a) (b) (c) (d) The protein binds to the small ribosomal subunit and increases the rate of initiation of translation. The protein binds to sequences in the 5′ region of the mRNA and inhibits the rate of initiation of translation. The protein binds to the large ribosomal subunit and slows down elongation of the polypeptide chain. The protein binds to sequences in the 3′ region of the mRNA and prevents termination of translation. 7-45 The concentration of a particular protein X in a normal human cell rises gradually from a low point, immediately after cell division, to a high point, just before cell division, and then drops sharply. The level of its mRNA in the cell remains fairly constant throughout this time. Protein X is required for cell growth and survival, but the drop in its level just before cell division is essential for division to proceed. You have isolated a line of human cells that grow in size in culture but cannot divide, and on analyzing these mutants, you find that levels of X mRNA in the mutant cells are normal. Which of the following mutations in the gene for X could explain these results? (a) the introduction of a stop codon that truncates protein X at the fourth amino acid (b) a change of the first ATG codon to CCA (c) the deletion of a sequence that encodes sites at which ubiquitin can be attached to the protein (d) a change at a splice site that prevents splicing of the RNA 7-46 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; use each word or phrase only once. Once an mRNA is produced, its message can be decoded on ribosomes. The ribosome is composed of two subunits: the __________________ subunit, which catalyzes the formation of the peptide bonds that link the amino acids together into a polypeptide chain, and the __________________ subunit, which matches the tRNAs to the codons of the mRNA. During the chain elongation process of translating an mRNA into protein, the growing polypeptide chain attached to a tRNA is bound to the __________________-site of the ribosome. An incoming aminoacyl-tRNA carrying the next amino acid in the chain will bind to the __________________-site by forming base pairs with the exposed codon in the mRNA. The __________________ enzyme catalyzes the formation of a new peptide bond between the growing polypeptide chain and the newly arriving amino acid. The end of a protein-coding message is signaled by the presence of a stop codon, which binds the __________________ called release factor. Eventually, most proteins will be degraded by a large complex of proteolytic enzymes called the __________________. A central DNA E large 7-47 medium P peptidyl transferase polymerase protein proteasome RNA small T ubiquitin After treating cells with a mutagen, you isolate two mutants. One carries alanine and the other carries methionine at a site in the protein that normally contains valine. After treating these two mutants again with mutagen, you isolate mutants from each that now carry threonine at the site of the original valine (see Figure Q7-47). Assuming that all mutations caused by the mutagen are due to single nucleotide changes, deduce the codons that are used for valine, alanine, methionine, and threonine at the affected site. (Refer to the codon table provided above Q7-29.) Figure Q7-47 7-48 Which of the following methods is not used by cells to regulate the amount of a protein in the cell? (a) Genes can be transcribed into mRNA with different efficiencies. (b) Many ribosomes can bind to a single mRNA molecule. (c) (d) 7-49 Proteins can be tagged with ubiquitin, marking them for degradation. Nuclear pore complexes can regulate the speed at which newly synthesized proteins are exported from the nucleus into the cytoplasm. Which of the following statements about the proteasome is false? (a) Ubiquitin is a small protein that is covalently attached to proteins to mark them for delivery to the proteasome. (b) Proteases reside in the central cylinder of a proteasome. (c) Misfolded proteins are delivered to the proteasome, where they are sequestered from the cytoplasm and can attempt to refold. (d) The protein stoppers that surround the central cylinder of the proteasome use the energy from ATP hydrolysis to move proteins into the proteasome inner chamber. RNA and the Origins of Life 7-50 Which of the following molecules is thought to have arisen first during evolution? (a) protein (b) DNA (c) RNA (d) All came to be at the same time. 7-51 According to current thinking, the minimum requirement for life to have originated on Earth was the formation of a _______________. (a) molecule that could provide a template for the production of a complementary molecule (b) double-stranded DNA helix (c) molecule that could direct protein synthesis (d) molecule that could catalyze its own replication 7-52 Ribozymes catalyze which of the following reactions? (a) DNA synthesis (b) transcription (c) RNA splicing (d) protein hydrolysis 7-53 You are studying a disease that is caused by a virus, but when you purify the virus particles and analyze them you find they contain no trace of DNA. Which of the following molecules are likely to contain the genetic information of the virus? (a) high-energy phosphate groups (b) RNA (c) lipids (d) carbohydrates 7-54 Give a reason why DNA makes a better material than RNA for the storage of genetic information, and explain your answer. How We Know: Cracking the Genetic Code 7-55 You have discovered an alien life form that surprisingly uses DNA as its genetic material, makes RNA from DNA, and reads the information from RNA to make protein using ribosomes and tRNAs, which read triplet codons. Because it is your job to decipher the genetic code for this alien, you synthesize some artificial RNA molecules and examine the protein products produced from these RNA molecules in a cell-free translation system using purified alien tRNAs and ribosomes. You obtain the results shown in Table Q7-55. Table Q7-55 From this information, which of the following peptides can be produced from poly UAUC? (a) Ile-Phe-Val-Tyr (b) Tyr-Ser-Phe-Ala (c) Ile-Lys-His-Tyr (d) Cys-Pro-Lys-Ala 7-56 An extraterrestrial organism (ET) is discovered whose basic cell biology seems pretty much the same as that of terrestrial organisms except that it uses a different genetic code to translate RNA into protein. You set out to break the code by translation experiments using RNAs of known sequence and cell-free extracts of ET cells to supply the necessary protein-synthesizing machinery. In experiments using the RNAs below, the following results were obtained when the 20 possible amino acids were added either singly or in different combinations of two or three: RNA 1: 5′-GCGCGCGCGCGCGCGCGCGCGCGCGCGC-3′ RNA 2: 5′-GCCGCCGCCGCCGCCGCCGCCGCCGCCGCC-3′ Using RNA 1, a polypeptide was produced only if alanine and valine were added to the reaction mixture. Using RNA 2, a polypeptide was produced only if leucine and serine and cysteine were added to the reaction mixture. Assuming that protein synthesis can start anywhere on the template, that the ET genetic code is nonoverlapping and linear, and that each codon is the same length (like the terrestrial triplet code), how many nucleotides does an ET codon contain? (a) 2 (b) 3 (c) 4 (d) 5 (e) 6 7-57 NASA has discovered an alien life form. You are called in to help NASA scientists to deduce the genetic code for this alien. Surprisingly, this alien life form shares many similarities with life on Earth: this alien uses DNA as its genetic material, makes RNA from DNA, and reads the information from RNA to make protein using ribosomes and tRNAs. Even more amazing, this alien uses the same 20 amino acids, like the organisms found on Earth, and also codes for each amino acid by a triplet codon. However, the scientists at NASA have found that the genetic code used by the alien life form differs from that used by life on Earth. NASA scientists drew this conclusion after creating a cell-free protein synthesis system from alien cells and adding an mRNA made entirely of uracil (poly U). They found that poly U directs the synthesis of a peptide containing only glycine. NASA scientists have synthesized a poly AU mRNA and observe that it codes for a polypeptide of alternating serine and proline amino acids. From these experiments, can you determine which codons code for serine and proline? Explain. Bonus question. Can you propose a mechanism for how the alien’s physiology is altered so that it uses a different genetic code from life on Earth, despite all the similarities?