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 rRNA transmembrane mRNA snRNA tRNA pRNA transcription proteins translation 7-4 Match the following structures with their names. 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 lac TFIIA CAP ligase TFIID deacetylase mediator TFIIH enhancer TATA 7-10 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) 5′-UAUCCGUAAGCUAGGCCUAUGCUA-3′ (b) 5′-AUAGGCAUUCGAUCCGGAUAGCAU-3′ (c) 5′-UACGAUAGGCCUAGCUUACGGAUA-3′ (d) none of the above 7-11 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) 5′- CCCUAAAAAAAAAAAAAAAAUUUUUUUUUUUUUUUUAGGG-3′ (b) 5′- UGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUG-3′ (c) 5′- AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3′ (d) 5′- GGAAAAGGAGAUGGGCAAGGGGAAAAGGAGAUGGGCAAGG-3′ 7-13 Imagine that an RNA polymerase is transcribing a segment of DNA that contains the following sequence: 5′-AGTCTAGGCACTGA-3′ 3′-TCAGATCCGTGACT 5′ A. If the polymerase is transcribing from this segment of DNA from left to right, which strand (top or bottom) is the template? B. What will be the sequence of that RNA (be sure to label the 5′ and 3′ ends of your RNA molecule)? 7-14 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) 7-methyl-G is added in a 5′ to 5′ linkage to the mRNA. (d) RNA polymerase dissociates. 7-20 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. transcription 2. translation 3. RNA splicing 4. polyadenylation 5. 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) 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. (c) Small RNA molecules in the nucleus perform the splicing reactions necessary for the removal of introns. (d) 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) Exons 2 and 3 must have the same number of nucleotides. (b) Exons 2 and 3 must contain an integral number of codons (that is, the number of nucleotides divided by 3 must be an integer). (c) 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). (d) 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. 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)? B. How many different peptides could potentially be made from this sequence of RNA, assuming that translation initiates upstream of this sequence? C. What are these peptides? (Give your answer using the one-letter amino acid code.) 7-36 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) a mutation in the DNA coding for the protein (b) a mutation in the anticodon of the isoleucine tRNA (tRNAIle) (c) a mutation in the isoleucyl-tRNA synthetase that decreases its ability to distinguish between different amino acids (d) 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. Match the labels given in the list below with the label lines in Figure Q7- 42. (a) ribosome-binding site (b) initiator codon (c) stop codon (d) untranslated 3′ region (e) untranslated 5′ region (f) protein-coding region B. 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) The protein binds to the small ribosomal subunit and increases the rate of initiation of translation. (b) The protein binds to sequences in the 5′ region of the mRNA and inhibits the rate of initiation of translation. (c) The protein binds to the large ribosomal subunit and slows down elongation of the polypeptide chain. (d) 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 medium proteasome central P RNA DNA peptidyl transferase small E polymerase T large protein ubiquitin 7-47 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) Proteins can be tagged with ubiquitin, marking them for degradation. (d) Nuclear pore complexes can regulate the speed at which newly synthesized proteins are exported from the nucleus into the cytoplasm. 7-49 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? CHAPTER 8 CONTROL OF GENE EXPRESSION 2009 Garland Science Publishing An Overview of Gene Expression 8-1 The distinct characteristics of different cell types in a multicellular organism result mainly from the differential regulation of the _________________. (a) replication of specific genes (b) transcription of genes transcribed by RNA polymerase II (c) transcription of housekeeping genes (d) packing of DNA into nucleosomes in some cells and not others 8-2 The human genome encodes about 24,000 genes. Approximately how many genes does the typical differentiated human cell express at any one time? (a) 24,000—all of them (b) between 21,500 and 24,000—at least 90% of the genes (c) between 5000 and 15,000 (d) less than 2500 8-3 Which of the following statements about differentiated cells is true? (a) Cells of distinct types express nonoverlapping sets of transcription factors. (b) Once a cell has differentiated, it can no longer change its gene expression. (c) Once a cell has differentiated, it will no longer need to transcribe RNA. (d) Some of the proteins found in differentiated cells are found in all cells of a multicellular organism. 8-4 Investigators performed nuclear transplant experiments to determine whether DNA is altered irreversibly during development. Which of the following statements about these experiments is true? (a) Because the donor nucleus is taken from an adult animal, the chromosomes from the nucleus must undergo recombination with the DNA in the egg for successful development to occur. (b) The embryo that develops from the nuclear transplant experiment is genetically identical to the donor of the nucleus. (c) The meiotic spindle of the egg must interact with the chromosomes of the injected nuclei for successful nuclear transplantation to occur. (d) Although nuclear transplantation has been successful in producing embryos in some mammals with the use of foster mothers, evidence of DNA alterations during differentiation has not been obtained for plants. 8-5 In principle, a eucaryotic cell can regulate gene expression at any step in the pathway from DNA to the active protein. Place the types of control listed below at the appropriate places on the diagram in Figure Q8-5. Figure Q8-5 A. translation control B. transcriptional control C. RNA processing control D. protein activity control How Transcriptional Switches Work 8-6 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. The genes of a bacterial __________________ are transcribed into a single mRNA. Many bacterial promoters contain a region known as a(n) __________________, to which a specific transcription regulator binds. Genes in which transcription is prevented are said to be __________________. The interaction of small molecules, such as tryptophan, with __________________ DNA-binding proteins, such as the tryptophan repressor, regulates bacterial genes. Genes that are being __________________ expressed are being transcribed all the time. allosteric negatively positively constitutively operator promoter induced operon repressed 8-7 Which of the following statements about transcriptional regulators is false? (a) Transcriptional regulators usually interact with the sugar-phosphate backbone on the outside of the double helix to determine where to bind on the DNA helix. (b) Transcriptional regulators will form hydrogen bonds, ionic bonds, and hydrophobic interactions with DNA. (c) The DNA-binding motifs of transcriptional regulators usually bind in the major groove of the DNA helix. (d) The binding of transcriptional regulators generally does not disrupt the hydrogen bonds that holds the double helix together. 8-8 Operons ___________________________. (a) are commonly found in eucaryotic cells (b) are transcribed by RNA polymerase II (c) contain a cluster of genes transcribed as a single mRNA (d) can only be regulated by gene activator proteins 8-9 The tryptophan operator ___________________________. (a) is an allosteric protein (b) binds to the tryptophan repressor when the repressor is bound to tryptophan (c) is required for production of the mRNA encoded by the tryptophan operon (d) is important for the production of the tryptophan repressor 8-10 Which of the following statements about the Lac operon is false? (a) The Lac repressor binds when lactose is present in the cell. (b) Even when the CAP activator is bound to DNA, if lactose is not present, the Lac operon will not be transcribed. (c) The CAP activator can only bind DNA when it is bound to cAMP. (d) The Lac operon only produces RNA when lactose is present and glucose is absent. 8-11 You are interested in examining the regulation of the gene that encodes an enzyme, Tre-ase, important in metabolizing trehalose into glucose in bacteria. Trehalose is a disaccharide formed of two glucose units. It is known that two DNA binding proteins, TreA and TreB, are important for binding to the promoter of the Tre-ase gene and are involved in regulating the transcription of the Tre-ase gene: TreA binds to the “A” site in the promoter region, and TreB binds to the “B” site. You make mutations in the TreA and TreB genes to create cells lacking these genes, observe what happens to transcription of the Tre-ase gene, and obtain the results in Table Q8-11. Table Q8-11 A. What is the role for TreA in controlling Tre-ase expression? Explain. B. What is the role for TreB in controlling Tre-ase expression? Explain. C. From these data, what do you predict will happen to Tre-ase transcription (compared with that in normal cells) in the presence of trehalose if you were to create a version of the TreA protein that will constitutively bind to the “A” site in the Tre-ase promoter? Note: Questions 8-12 to 8-15 use the following information and the data in Table Q8-12. These questions may be used independently, or as a group. You are interested in examining the Psf gene. It is known that Psf is normally produced when cells are exposed to high levels of both calcium (Ca2+) and magnesium (Mg2+). MetA, MetB, and MetC are important for binding to the promoter of the Psf gene and are involved in regulating its transcription. MetA binds to the “A” site in the promoter region, MetB to the “B” site, and MetC to the “C” site. You create binding site mutations in the A, B, and C sites and observe what happens to transcription of the Psf gene. Your results are summarized in Table Q8-12. Table Q8-12 For this table: -, no transcription of Psf; +, low level of transcription of Psf; ++, high levels of transcription of Psf. 8-12 Which of the following proteins are likely to act as gene activators? (a) MetA only (b) MetB only (c) MetC only (d) Both MetA and MetC 8-13 Which of the following proteins are likely to act as gene repressors? (a) MetA only (b) MetB only (c) MetC only (d) Both MetA and MetC 8-14 Which transcription factors are normally bound to the Psf promoter in the presence of Mg2+ only? (a) none (b) MetA only (c) MetA and Met B (d) MetA, MetB, and MetC 8-15 Which transcription factors are normally bound to the Psf promoter in the presence of both Mg2+ and Ca2+? (a) MetA and MetB (b) MetB and MetC (c) MetA and MetC (d) MetA, MetB, and MetC Note: Questions 8-16 to 8-19 use the following information and the data in Figure Q8-16. These questions may be used independently, or as a group. You are interested in understanding the gene regulation of Lkp1, a protein that is normally produced in liver and kidney cells in mice. Interestingly, you find that the LKP1 gene is not expressed in heart cells. You isolate the DNA upstream of the LKP1 gene, place it upstream of the gene for green fluorescent protein (GFP), and insert this entire piece of recombinant DNA into mice. You find GFP expressed in liver and kidney cells but not in heart cells, an expression pattern similar to the normal expression of the LKP1 gene. Further experiments demonstrate that there are three regions in the promoter, labeled A, B, and C in Figure Q8-16, that contribute to this expression pattern. Assume that a single and unique transcription factor binds each site such that protein X binds site A, protein Y binds site B, and protein Z binds site C. You want to determine which region is responsible for tissue-specific expression, and create mutations in the promoter to determine the function of each of these regions. In Figure Q8-16, if the site is missing, it is mutated such that it cannot bind its corresponding transcription factor. Figure Q8-16 8-16 Which of the following proteins are likely to act as gene repressors? (a) factor X (b) factor Y (c) factor Z (d) none of the above 8-17 Which of the following proteins are likely to act as gene activators? (a) factors X and Y (b) factors X and Z (c) factors Y and Z (d) factor X only 8-18 Experiment 1 in Figure Q8-16 is the positive control, demonstrating that the region of DNA upstream of the gene for GFP results in a pattern of expression that we normally find for the LKP1 gene. Experiment 2 shows what happens when the sites for binding factors X, Y, and Z are removed. Which experiment above demonstrates that factor X alone is sufficient for expression of LPK1 in the kidney? (a) experiment 3 (b) experiment 5 (c) experiment 6 (d) experiment 7 8-19 In what tissue is factor Z normally present and bound to the DNA? (a) kidney (b) liver (c) heart (d) none of the above 8-20 An allosteric transcription regulator called HisP regulates the enzymes for histidine biosynthesis in the bacterium E. coli. Histidine modulates HisP activity. On binding histidine, HisP alters its conformation, markedly changing its affinity for the regulatory sequences in the promoters of the genes for the histidine biosynthetic enzymes. A. If HisP functions as a gene repressor, would you expect that HisP would bind more tightly or less tightly to the regulatory sequences when histidine is abundant? Explain your answer. B. If HisP functions as a gene activator, would you expect that HisP would bind more tightly or less tightly to the regulatory sequences when histidine levels are low? Explain your answer. 8-21 Bacterial cells can take up the amino acid tryptophan from their surroundings, or, if the external supply is insufficient, they can synthesize trytophan by using enzymes in the cell. In some bacteria, the control of glutamine synthesis is similar to that of tryptophan synthesis, such that the glutamine repressor inhibits the transcription of the glutamine operon, which contains the genes that code for the enzymes required for glutamine synthesis. On binding to cellular glutamine, the glutamine repressor binds to a site in the promoter of the operon. A. Why is glutamine-dependent binding to the operon a useful property for the glutamine repressor? B. What would you expect to happen to the regulation of the enzymes that synthesize glutamine in cells expressing a mutant form of the glutamine repressor that cannot bind to DNA? C. What would you expect to happen to the regulation of the enzymes that synthesize glutamine in cells expressing a mutant form of the glutamine repressor that binds to DNA even when no glutamine is bound to it? 8-22 In the absence of glucose, E. coli can proliferate by using the pentose sugar arabinose. As shown in Figure Q8-22, the arabinose operon regulates the ability of E. coli to use arabinose. The araA, araB, and araD genes encode enzymes for the metabolism of arabinose. The araC gene encodes a transcription regulator that binds adjacent to the promoter of the arabinose operon. To understand the regulatory properties of the AraC protein, you engineer a mutant bacterium in which the araC gene has been deleted and look at the effect of the presence or absence of the AraC protein on the AraA enzyme. Figure Q8-22 A. If the AraC protein works as a gene repressor, would you expect araA RNA levels to be high or low in the presence of arabinose in the araCmutant cells? What about in the araC- mutant cells in the absence of arabinose? Explain your answer. B. Your findings from the experiment are summarized in Table Q8-22. Table Q8-22 Do the results in Table Q8-22 indicate that the AraC protein regulates arabinose metabolism by acting as a gene repressor or a gene activator? Explain your answer. 8-23 The CAP activator protein and the Lac repressor both control the Lac operon (see Figure Q8-23). You create cells that are mutant in the gene coding for the Lac repressor so that these cells lack the Lac repressor under all conditions. For these mutant cells, state whether the Lac operon will be switched on or off in the following situations and explain why. Figure Q8-23 A. in the presence of glucose and lactose B. in the presence of glucose and the absence of lactose C. in the absence of glucose and the absence of lactose D. in the absence of glucose and the presence of lactose 8-24 You have discovered an operon in a bacterium that is turned on only when sucrose is present and glucose is absent. You have also isolated three mutants that have changes in the upstream regulatory sequences of the operon and whose behavior is summarized in the Table Q8-24. You hypothesize that there are two gene regulatory sites in the upstream regulatory sequence, A and B, which are affected by the mutations. For this question, a plus (+) indicates a normal site and a minus (-) indicates a mutant site that no longer binds its transcription regulator. Table Q8-24 A. If mutant 1 has sites A- B+ , which of these sites is regulated by sucrose and which by glucose? B. Give the state (+ or -) of the A and B sites in mutants 2 and 3. C. Which site is bound by a repressor and which by an activator? 8-25 Label the following structures in Figure Q8-25. Figure Q8-25 A. activator protein B. RNA polymerase C. general transcription factors D. mediator 8-26 How are most eucaryotic transcription regulators able to affect transcription when their binding sites are far from the promoter? (a) by binding to their binding site and sliding to the site of RNA polymerase assembly (b) by looping out the intervening DNA between their binding site and the promoter (c) by unwinding the DNA between their binding site and the promoter (d) by attracting RNA polymerase and modifying it before it can bind to the promoter 8-27 The expression of the BRF1 gene in mice is normally quite low, but mutations in a gene called BRF2 lead to increased expression of BRF1. You have a hunch that nucleosomes are involved in the regulation of BRF1 expression and so you investigate the position of nucleosomes over the TATA box of BRF1 in normal mice and in mice that lack either the BRF2 protein (BRF2- ) or part of histone H4 (HHF- ) (histone H4 is encoded by the HHF gene). Table Q8-27 summarizes your results. A normal functional gene is indicated by a plus sign (+). Table Q8-27 Which of the following conclusions cannot be drawn from your data? Explain your answer. (a) BRF2 is required for the repression of BRF1. (b) BRF2 is required for the specific pattern of nucleosome positions over the BRF1 upstream region. (c) The specific pattern of nucleosome positioning over the BRF1 upstream region is required for BRF1 repression. (d) The part of histone H4 missing in HHF- mice is not required for the formation of nucleosomes. 8-28 The yeast GAL4 gene encodes a transcriptional regulator that can bind DNA upstream of genes required for the metabolism of the sugar galactose and turns them on. Gal4 has a DNA-binding domain and an activation domain. The DNAbinding domain allows it to bind to the appropriate sites in the promoters of the galactose metabolism genes. The activation domain attracts histone-modifying enzymes and also binds to a component of the RNA polymerase II enzyme complex, attracting it to the promoter so that the regulated genes can be turned on when Gal4 is also bound to the DNA. When Gal4 is expressed normally, the genes can be maximally activated. You decide to try to produce more of the galactose metabolism genes by overexpressing the Gal4 protein at levels fiftyfold greater than normal. You conduct experiments to show that you are overexpressing the Gal4 protein and that it is properly localized in the nucleus of the yeast cells. To your surprise, you find that too much Gal4 causes the galactose genes to be transcribed only at a low level. What is the most likely explanation for your findings? 8-29 For each of the following sentences, fill in the blanks with the best word or phrase in the list below. Not all words or phrases will be used; use each word or phrase only once. During transcription in __________________ cells, transcriptional regulators that bind to DNA thousands of nucleotides away from a gene’s promoter can affect a gene’s transcription. The __________________ is a complex of proteins that links distantly bound transcription regulators with the proteins bound closer to the transcriptional start site. Transcriptional activators can also interact with histone __________________s, which alter chromatin by modifying lysines in the tail of histone proteins to allow greater accessibility to the underlying DNA. Gene repressor proteins can reduce the efficiency of transcription initiation by attracting histone __________________s. Sometimes, many contiguous genes can become transcriptionally inactive as a result of chromatin remodeling, like the __________________ found in interphase chromosomes. viral acetylase centrosome helicase eucaryotic operator procaryotic peroxidase deacetylase mediator deoxidase heterochromatin telomere enhancer leucine zipper The Molecular Mechanisms that Create Specialized Cell Types 8-30 In principle, how many different cell types can an organism having four different types of transcription regulator and thousands of genes create? (a) up to 4 (b) up to 8 (c) up to 16 (d) thousands 8-31 From the sequencing of the human genome, we believe that there are approximately 24,000 protein-coding genes in the genome, for which there are an estimated 1500–3000 transcription factors. If every gene has a tissue-specific and signal-dependent transcription pattern, how can such a small number of transcriptional regulatory proteins generate a much larger set of transcriptional patterns? 8-32 Combinatorial control of gene expression __________________________. (a) involves every gene using a different combination of transcriptional regulators for its proper expression (b) involves groups of transcriptional regulators working together to determine the expression of a gene (c) involves only the use of gene activators used together to regulate genes appropriately (d) is seen only when genes are arranged in operons 8-33 You are studying a set of mouse genes whose expression increases when cells are exposed to the hormone cortisol, and you believe that the same cortisolresponsive transcriptional activator regulates all of these genes. Which of the following statements below should be true if your hypothesis is correct? (a) The cortisol-responsive genes share a DNA sequence in their regulatory regions that binds the cortisol-responsive transcriptional activator. (b) The cortisol-responsive genes must all be in an operon. (c) The transcriptional regulators that bind to the regulatory regions of the cortisol-responsive genes must all be the same. (d) The cortisol-responsive genes must not be transcribed in response to other hormones. 8-34 The MyoD transcriptional regulator is normally found in differentiating muscle cells and participates in the transcription of genes that produce muscle-specific proteins, such as those needed in contractile tissue. Amazingly, expression of MyoD in fibroblasts causes these cells derived from skin connective tissue to produce proteins normally only seen in muscles. However, some other cell types do not transcribe muscle-specific genes when MyoD is expressed in them. Which of the following statements below is the best explanation of why MyoD can cause fibroblasts to express muscle-specific genes? (a) Unlike some other cell types, fibroblasts have not lost the muscle-specific genes from their genome. (b) The muscle-specific genes must be in heterochromatin in fibroblasts. (c) During their developmental history, fibroblasts have accumulated some transcriptional regulators in common with differentiating muscle cells. (d) The presence of MyoD is sufficient to activate the transcription of musclespecific genes in all cell types. 8-35 A virus produces a protein X that activates only a few of the virus’s own genes (V1, V2, and V3) when it infects cells. The cellular proteins A (a zinc finger protein) and the cellular protein B (a homeodomain protein) are known to be repressors of the viral genes V1, V2, and V3. You examine the complete upstream gene regulatory sequences of these three viral genes and find the following: 1. V1 and V2 contain binding sites for the zinc finger protein A only. 2. V3 contains a binding site for the homeodomain protein B only. 3. The only sequence that all three genes have in common is the TATA box. Label each of the choices below as likely or unlikely as an explanation for your findings. For each choice you label as unlikely, explain why. A. Protein X binds nonspecifically to the DNA upstream of V1, V2, and V3 and activates transcription. B. Protein X binds to a repressor and prevents the repressor from binding upstream of V1, V2, and V3. C. Protein X activates transcription by binding to the TATA box. D. Protein X activates transcription by binding to and sequestering proteins A and B. E. Protein X represses transcription of the genes for proteins A and B. 8-36 In mammals, individuals with two X chromosomes are female, and individuals with an X and a Y chromosome are male. It had long been known that a gene located on the Y chromosome was sufficient to induce the gonads to form testes, which is the main male-determining factor in development, and researchers sought the product of this gene, the so-called testes-determining factor (TDF). For several years, the TDF was incorrectly thought to be a zinc finger protein encoded by a gene called BoY. Which of the following observations would most strongly suggest that BoY might not be the TDF? Explain your answer. (a) Some XY individuals that develop into females have mutations in a different gene, SRY, but are normal at BoY. (b) BoY is not expressed in the adult male testes. (c) Expression of BoY in adult females does not masculinize them. (d) A few of the genes that are known to be expressed only in the testes have binding sites for the BoY protein in their upstream regulatory sequences, but most do not. 8-37 Which of the following is not a general mechanism that cells use to maintain stable patterns of gene expression as cells divide? (a) a positive feedback loop, mediated by a transcriptional regulator that activates transcription of its own gene in addition to other cell-type specific genes (b) faithful propagation of condensed chromatin structures as cells divide (c) inheritance of DNA methylation patterns when cells divide (d) proper segregation of housekeeping proteins when cells divide 8-38 Which of the following statements about DNA methylation in eucaryotes is false? (a) Appropriate inheritance of DNA methylation patterns involves maintenance methyltransferase. (b) DNA methylation involves a covalent modification of cytosine bases. (c) Methylation of DNA attracts proteins that block gene expression. (d) Immediately after DNA replication, each daughter helix contains one methylated DNA strand, which corresponds to the newly synthesized strand. 8-39 Which of the following statements about the Ey transcriptional regulator is false? (a) Expression of Ey in cells that normally form legs in the fly will lead to the formation of an eye in the middle of the legs. (b) The Ey transcription factor must bind to the promoter of every eyespecific gene in the fly. (c) Positive feedback loops ensure that Ey expression remains switched on in the developing eye. (d) A homolog of Ey is found in vertebrates; this homolog is also used during eye development. Post-Transcriptional Controls 8-40 Which of the following statements about riboswitches is false? (a) Riboswitches can block the production of mRNAs. (b) Riboswitches can control the translation of mRNAs. (c) Riboswitches are made from rRNAs. (d) Riboswitches can bind metabolites. 8-41 Which of the following is not involved in post-transcriptional control? (a) the spliceosome (b) dicer (c) mediator (d) RISC 8-42 MicroRNAs ____________________. (a) are produced from a precursor miRNA transcript (b) are found only in humans (c) control gene expression by base-pairing with DNA sequences (d) can degrade RNAs by using their intrinsic catalytic activity 8-43 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. MicroRNAs are noncoding RNAs that are incorporated into a protein complex called __________________, which searches the __________________s in the cytoplasm for sequence complementary to that of the miRNA. When such a molecule is found, it is then targeted for __________________. RNAi is triggered by the presence of foreign __________________ molecules, which are digested by the __________________ enzyme into shorter fragments approximately 23 nucleotide pairs in length. tRNA phosphorylation riboswitch RISC destruction acetylation procaryotic methylation DNA double-stranded RNA mRNA dicer single-stranded RNA rRNA mitochondria 8-44 The extent of complementarity of a miRNA with its target mRNA determines ___________________________. (a) whether the mRNA will be degraded or transported elsewhere in the cell (b) whether the mRNA will be transported to the nucleus (c) whether RISC is degraded (d) whether the miRNA synthesizes a complementary strand 8-45 Which of the following statements about miRNAs is false? (a) One miRNA can regulate the expression of many genes. (b) miRNAs are transcribed in the nucleus from genomic DNA. (c) miRNAs are produced from rRNAs. (d) miRNAs are made by RNA polymerase. 8-46 Which of the following statements about RNAi is true? (a) The RNAi mechanism is found only in plants and animals. (b) RNAi is induced when double-stranded RNA is present in the cell. (c) RISC uses the siRNA duplex to locate complementary foreign RNA molecules. (d) siRNAs bind to miRNAs to induce RNAi. How We Know: Gene Regulation – The Story of Eve 8-47 The gene for a hormone necessary for insect development contains binding sites for three transcription regulators called A, B, and C. Because the binding sites for A and B overlap, A and B cannot bind simultaneously. You make mutations in the binding sites for each of the proteins and measure hormone production in cells that contain equal amounts of the A, B, and C proteins. Figure Q8-47 summarizes your results. In each of the following sentences, choose one of the phrases within square brackets to make the statement consistent with the results. Figure Q8-47 A. Protein A binds to its DNA-binding site [more tightly/less tightly] than protein B binds to its DNA-binding site. B. Protein A is a [stronger/weaker] activator of transcription than protein B. C. Protein C is able to prevent activation by [protein A only/protein B only/both protein A and protein B]. 8-48 The Drosophila Eve gene has a complex promoter containing multiple binding sites for four transcription regulators: Bicoid, Hunchback, Giant, and Krüppel. Bicoid and Hunchback are activators of Eve transcription, whereas Giant and Krüppel repress Eve transcription. Figure Q8-48A shows the patterns of expression of these regulators. Figure Q8-48 The eve promoter contains modules that control expression in various stripes. You construct a reporter gene that contains the DNA 5 kb upstream of the eve gene, so that this reporter contains the stripe 3 module, the stripe 2 module, the stripe 7 module, and the TATA box, all fused to the LacZ reporter gene (which encodes the β-galactosidase enzyme), as shown in Figure Q8-50B. This construct results in expression of the β-galactosidase enzyme in three stripes, which correspond to the normal positions of stripes 3, 2, and 7. A. By examining the overlap of sites on the stripe 2 module, as depicted in Figure Q8-48B, what is the biological effect of having some of the transcription regulator binding sites overlap? B. You make two mutant versions in which several of the binding sites in the Eve stripe 2 module have been deleted, as detailed in items (i) and (ii) below. Refer to Figure Q8-48B for the positions of the binding sites. (Note, however, that because many of the binding sites overlap, it is not possible to delete all of one kind of site without affecting some of the other sites.) Match the appropriate mutant condition with the most likely pattern of Eve expression shown in Figure Q8-48C. Explain your choices. (i) deletion of the Krüppel-binding sites in stripe 2 (ii) deletion of the two Bicoid-binding sites in the stripe 2 module that are marked with an asterisk (*) in Figure Q8-48B
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