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Biochemistry_Lippincott_1483	Biochemistry_Lippinco	A. Mismatch repair Sometimes replication errors escape the proofreading activity during DNA synthesis, causing a mismatch of one to several bases. In E. coli, mismatch repair (MMR) is mediated by a group of proteins known as the Mut proteins (Fig. 30.28). Homologous proteins are present in humans. [Note: MMR occurs within minutes of replication and reduces the error rate of replication from 1 in 107 to 1 in 109 nucleotides.] 1.	Biochemistry_Lippinco. A. Mismatch repair Sometimes replication errors escape the proofreading activity during DNA synthesis, causing a mismatch of one to several bases. In E. coli, mismatch repair (MMR) is mediated by a group of proteins known as the Mut proteins (Fig. 30.28). Homologous proteins are present in humans. [Note: MMR occurs within minutes of replication and reduces the error rate of replication from 1 in 107 to 1 in 109 nucleotides.] 1.
Biochemistry_Lippincott_1484	Biochemistry_Lippinco	Mismatched strand identification: When a mismatch occurs, the Mut proteins that identify the mispaired nucleotide(s) must be able to discriminate between the correct strand and the strand with the mismatch. In prokaryotes, discrimination is based on the degree of methylation. GATC sequences, which are found once every thousand nucleotides, are methylated on the adenine (A) residue by DNA adenine methylase (DAM). This methylation is not done immediately after synthesis, so the DNA is hemimethylated (that is, the parental strand is methylated, but the daughter strand is not). The methylated parental strand is assumed to be correct, and it is the daughter strand that gets repaired. [Note: The exact mechanism by which the daughter strand is identified in eukaryotes is not yet known, but likely involves recognition of nicks in the newly synthesized strand.] 2.	Biochemistry_Lippinco. Mismatched strand identification: When a mismatch occurs, the Mut proteins that identify the mispaired nucleotide(s) must be able to discriminate between the correct strand and the strand with the mismatch. In prokaryotes, discrimination is based on the degree of methylation. GATC sequences, which are found once every thousand nucleotides, are methylated on the adenine (A) residue by DNA adenine methylase (DAM). This methylation is not done immediately after synthesis, so the DNA is hemimethylated (that is, the parental strand is methylated, but the daughter strand is not). The methylated parental strand is assumed to be correct, and it is the daughter strand that gets repaired. [Note: The exact mechanism by which the daughter strand is identified in eukaryotes is not yet known, but likely involves recognition of nicks in the newly synthesized strand.] 2.
Biochemistry_Lippincott_1485	Biochemistry_Lippinco	Repair procedure: When the strand containing the mismatch is identified, an endonuclease nicks the strand, and the mismatched nucleotide(s) is/are removed by an exonuclease. Additional nucleotides at the 5′-and 3′-ends of the mismatch are also removed. The gap left by removal of the nucleotides is filled, using the sister strand as a template, by a DNA pol, typically DNA pol III. The 3′-hydroxyl of the newly synthesized DNA is joined to the 5′-phosphate of the remaining stretch of the original DNA strand by DNA ligase. Mutation to the proteins involved in MMR in humans is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. Although HNPCC confers an increased risk for developing colon cancer (as well as other cancers), only about 5% of all colon cancer is the result of mutations in MMR. B. Nucleotide excision repair	Biochemistry_Lippinco. Repair procedure: When the strand containing the mismatch is identified, an endonuclease nicks the strand, and the mismatched nucleotide(s) is/are removed by an exonuclease. Additional nucleotides at the 5′-and 3′-ends of the mismatch are also removed. The gap left by removal of the nucleotides is filled, using the sister strand as a template, by a DNA pol, typically DNA pol III. The 3′-hydroxyl of the newly synthesized DNA is joined to the 5′-phosphate of the remaining stretch of the original DNA strand by DNA ligase. Mutation to the proteins involved in MMR in humans is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. Although HNPCC confers an increased risk for developing colon cancer (as well as other cancers), only about 5% of all colon cancer is the result of mutations in MMR. B. Nucleotide excision repair
Biochemistry_Lippincott_1486	Biochemistry_Lippinco	B. Nucleotide excision repair Exposure of a cell to UV radiation can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. These intrastrand cross-links prevent DNA pol from replicating the DNA strand beyond the site of dimer formation. Thymine dimers are excised in bacteria by UvrABC proteins in a process known as nucleotide excision repair (NER), as illustrated in Figure 30.29. A related pathway is present in humans (see 2. below). [Note: Transcription-coupled repair, a type of NER, fixes DNA lesions encountered during RNA synthesis.] 1. Recognition and excision of UV-induced dimers: A UV-specific endonuclease (called uvrABC excinuclease) recognizes the bulky dimer and cleaves the damaged strand on both the 5′-side and 3′-side of the lesion. A short oligonucleotide containing the dimer is excised, leaving a gap in the DNA strand. This gap is filled in using a DNA pol I and DNA ligase. NER occurs throughout the cell cycle. 2.	Biochemistry_Lippinco. B. Nucleotide excision repair Exposure of a cell to UV radiation can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. These intrastrand cross-links prevent DNA pol from replicating the DNA strand beyond the site of dimer formation. Thymine dimers are excised in bacteria by UvrABC proteins in a process known as nucleotide excision repair (NER), as illustrated in Figure 30.29. A related pathway is present in humans (see 2. below). [Note: Transcription-coupled repair, a type of NER, fixes DNA lesions encountered during RNA synthesis.] 1. Recognition and excision of UV-induced dimers: A UV-specific endonuclease (called uvrABC excinuclease) recognizes the bulky dimer and cleaves the damaged strand on both the 5′-side and 3′-side of the lesion. A short oligonucleotide containing the dimer is excised, leaving a gap in the DNA strand. This gap is filled in using a DNA pol I and DNA ligase. NER occurs throughout the cell cycle. 2.
Biochemistry_Lippincott_1487	Biochemistry_Lippinco	2. UV radiation and cancer: Pyrimidine dimers can be formed in the skin cells of humans exposed to UV radiation in unfiltered sunlight. In the rare genetic disease xeroderma pigmentosum (XP), the cells cannot repair the damaged DNA, resulting in extensive accumulation of mutations and, consequently, early and numerous skin cancers (Fig. 30.30). XP can be caused by defects in any of the several genes that code for the XP proteins required for NER of UV damage in humans. C. Base excision repair	Biochemistry_Lippinco. 2. UV radiation and cancer: Pyrimidine dimers can be formed in the skin cells of humans exposed to UV radiation in unfiltered sunlight. In the rare genetic disease xeroderma pigmentosum (XP), the cells cannot repair the damaged DNA, resulting in extensive accumulation of mutations and, consequently, early and numerous skin cancers (Fig. 30.30). XP can be caused by defects in any of the several genes that code for the XP proteins required for NER of UV damage in humans. C. Base excision repair
Biochemistry_Lippincott_1488	Biochemistry_Lippinco	C. Base excision repair DNA bases can be altered, either spontaneously, as is the case with cytosine, which slowly undergoes deamination (the loss of its amino group) to form uracil, or by the action of deaminating or alkylating compounds. For example, nitrous acid, which is formed by the cell from precursors such as the nitrates, deaminates cytosine, adenine (to hypoxanthine), and guanine (to xanthine). Dimethyl sulfate can alkylate (methylate) adenine. Bases can also be lost spontaneously. For example, ~10,000 purine bases are lost this way per cell per day. Lesions involving base alterations or loss can be corrected by base excision repair ([BER], Fig. 30.31). 1.	Biochemistry_Lippinco. C. Base excision repair DNA bases can be altered, either spontaneously, as is the case with cytosine, which slowly undergoes deamination (the loss of its amino group) to form uracil, or by the action of deaminating or alkylating compounds. For example, nitrous acid, which is formed by the cell from precursors such as the nitrates, deaminates cytosine, adenine (to hypoxanthine), and guanine (to xanthine). Dimethyl sulfate can alkylate (methylate) adenine. Bases can also be lost spontaneously. For example, ~10,000 purine bases are lost this way per cell per day. Lesions involving base alterations or loss can be corrected by base excision repair ([BER], Fig. 30.31). 1.
Biochemistry_Lippincott_1489	Biochemistry_Lippinco	1. Abnormal base removal: In BER, abnormal bases, such as uracil, which can occur in DNA by either deamination of cytosine or improper use of dUTP instead of dTTP during DNA synthesis, are recognized by specific DNA glycosylases that hydrolytically cleave them from the deoxyribosephosphate backbone of the strand. This leaves an apyrimidinic site, or apurinic if a purine was removed, both referred to as AP sites. 2. AP site recognition and repair: Specific AP endonucleases recognize that a base is missing and initiate the process of excision and gap filling by making an endonucleolytic cut just to the 5′-side of the AP site. A deoxyribose phosphate lyase removes the single, base-free, sugar phosphate residue. DNA pol I and DNA ligase complete the repair process. D. Double-strand break repair	Biochemistry_Lippinco. 1. Abnormal base removal: In BER, abnormal bases, such as uracil, which can occur in DNA by either deamination of cytosine or improper use of dUTP instead of dTTP during DNA synthesis, are recognized by specific DNA glycosylases that hydrolytically cleave them from the deoxyribosephosphate backbone of the strand. This leaves an apyrimidinic site, or apurinic if a purine was removed, both referred to as AP sites. 2. AP site recognition and repair: Specific AP endonucleases recognize that a base is missing and initiate the process of excision and gap filling by making an endonucleolytic cut just to the 5′-side of the AP site. A deoxyribose phosphate lyase removes the single, base-free, sugar phosphate residue. DNA pol I and DNA ligase complete the repair process. D. Double-strand break repair
Biochemistry_Lippincott_1490	Biochemistry_Lippinco	Ionizing radiation, chemotherapeutic agents such as doxorubicin, and oxidative free radicals (see p. 148) can cause double-strand breaks in DNA that can be lethal to the cell. [Note: Such breaks also occur naturally during genetic recombination.] dsDNA breaks cannot be corrected by the previously described strategy of excising the damage on one strand and using the undamaged strand as a template for replacing the missing nucleotide(s). Instead, they are repaired by one of two systems. The first is nonhomologous end joining (NHEJ), in which a group of proteins mediates the recognition, processing, and ligation of the ends of two DNA fragments. However, some DNA is lost in the process. Consequently, NHEJ is error prone and mutagenic. Defects in NHEJ are associated with a predisposition to cancer and immunodeficiency syndromes. The second repair system, homologous recombination (HR), uses the enzymes that normally perform genetic recombination between homologous chromosomes during	Biochemistry_Lippinco. Ionizing radiation, chemotherapeutic agents such as doxorubicin, and oxidative free radicals (see p. 148) can cause double-strand breaks in DNA that can be lethal to the cell. [Note: Such breaks also occur naturally during genetic recombination.] dsDNA breaks cannot be corrected by the previously described strategy of excising the damage on one strand and using the undamaged strand as a template for replacing the missing nucleotide(s). Instead, they are repaired by one of two systems. The first is nonhomologous end joining (NHEJ), in which a group of proteins mediates the recognition, processing, and ligation of the ends of two DNA fragments. However, some DNA is lost in the process. Consequently, NHEJ is error prone and mutagenic. Defects in NHEJ are associated with a predisposition to cancer and immunodeficiency syndromes. The second repair system, homologous recombination (HR), uses the enzymes that normally perform genetic recombination between homologous chromosomes during
Biochemistry_Lippincott_1491	Biochemistry_Lippinco	to cancer and immunodeficiency syndromes. The second repair system, homologous recombination (HR), uses the enzymes that normally perform genetic recombination between homologous chromosomes during meiosis. This system is much less error prone (“error-free”) than NHEJ because any DNA that was lost is replaced using homologous DNA as a template. HR occurs in late S and G2 of the cell cycle, whereas NHEJ can occur anytime.	Biochemistry_Lippinco. to cancer and immunodeficiency syndromes. The second repair system, homologous recombination (HR), uses the enzymes that normally perform genetic recombination between homologous chromosomes during meiosis. This system is much less error prone (“error-free”) than NHEJ because any DNA that was lost is replaced using homologous DNA as a template. HR occurs in late S and G2 of the cell cycle, whereas NHEJ can occur anytime.
Biochemistry_Lippincott_1492	Biochemistry_Lippinco	[Note: Mutations to the proteins BRCA1 or BRCA2 (breast cancer 1 or 2), which are involved in HR, increase the risk for developing breast and ovarian cancer.] VII. CHAPTER SUMMARY	Biochemistry_Lippinco. [Note: Mutations to the proteins BRCA1 or BRCA2 (breast cancer 1 or 2), which are involved in HR, increase the risk for developing breast and ovarian cancer.] VII. CHAPTER SUMMARY
Biochemistry_Lippincott_1493	Biochemistry_Lippinco	DNA is a polymer of deoxynucleoside monophosphates covalently linked by 3′→5′-phosphodiester bonds (Fig. 30.32). The resulting long, unbranched chain has polarity, with both a 5′-end (free phosphate) and a 3′end (free hydroxyl). The sequence of nucleotides is read 5′→3′. DNA exists as a double-stranded molecule, in which the two chains are paired in an antiparallel manner and wind around each other, forming a double helix. Adenine pairs with thymine, and cytosine pairs with guanine. Each strand of the double helix serves as a template for constructing a complementary daughter strand (semiconservative replication). DNA replication occurs in the S phase of the cell cycle and begins at an origin of replication. As the two strands unwind and separate, synthesis occurs at two replication forks that move away from the origin in opposite directions (bidirectionally). Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in	Biochemistry_Lippinco. DNA is a polymer of deoxynucleoside monophosphates covalently linked by 3′→5′-phosphodiester bonds (Fig. 30.32). The resulting long, unbranched chain has polarity, with both a 5′-end (free phosphate) and a 3′end (free hydroxyl). The sequence of nucleotides is read 5′→3′. DNA exists as a double-stranded molecule, in which the two chains are paired in an antiparallel manner and wind around each other, forming a double helix. Adenine pairs with thymine, and cytosine pairs with guanine. Each strand of the double helix serves as a template for constructing a complementary daughter strand (semiconservative replication). DNA replication occurs in the S phase of the cell cycle and begins at an origin of replication. As the two strands unwind and separate, synthesis occurs at two replication forks that move away from the origin in opposite directions (bidirectionally). Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in
Biochemistry_Lippincott_1494	Biochemistry_Lippinco	that move away from the origin in opposite directions (bidirectionally). Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in the region of DNA ahead of the replication fork and negative supercoils behind the fork. DNA topoisomerases types I and II remove supercoils. DNA polymerases (pols) synthesize new DNA strands only in the 5′→3′ direction. Therefore, one of the newly synthesized stretches of nucleotide chains must grow in the 5′→3′ direction toward the replication fork (leading strand) and one in the 5′→3′ direction away from the replication fork (lagging strand). DNA pols require a primer, a short stretch of RNA synthesized by primase. Leading-strand synthesis needs only one RNA primer (continuous synthesis), whereas the lagging strand needs many (discontinuous synthesis involving Okazaki fragments). In Escherichia coli (E. coli), DNA chain elongation is catalyzed by DNA pol III, using 5′-deoxyribonucleoside	Biochemistry_Lippinco. that move away from the origin in opposite directions (bidirectionally). Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in the region of DNA ahead of the replication fork and negative supercoils behind the fork. DNA topoisomerases types I and II remove supercoils. DNA polymerases (pols) synthesize new DNA strands only in the 5′→3′ direction. Therefore, one of the newly synthesized stretches of nucleotide chains must grow in the 5′→3′ direction toward the replication fork (leading strand) and one in the 5′→3′ direction away from the replication fork (lagging strand). DNA pols require a primer, a short stretch of RNA synthesized by primase. Leading-strand synthesis needs only one RNA primer (continuous synthesis), whereas the lagging strand needs many (discontinuous synthesis involving Okazaki fragments). In Escherichia coli (E. coli), DNA chain elongation is catalyzed by DNA pol III, using 5′-deoxyribonucleoside
Biochemistry_Lippincott_1495	Biochemistry_Lippinco	the lagging strand needs many (discontinuous synthesis involving Okazaki fragments). In Escherichia coli (E. coli), DNA chain elongation is catalyzed by DNA pol III, using 5′-deoxyribonucleoside triphosphates as substrates. The enzyme proofreads the newly synthesized DNA, removing terminal mismatched nucleotides with its 3′→5′ exonuclease activity. RNA primers are removed by DNA pol I, using its 5′→3′ exonuclease activity. This enzyme fills the gaps with DNA, proofreading as it synthesizes. The final phosphodiester linkage is catalyzed by DNA ligase. There are at least five high-fidelity eukaryotic DNA pols. Pol α is a multisubunit enzyme, one subunit of which is a primase. Pol α 5′→3′ polymerase activity adds a short piece of DNA to the RNA primer. Pol ε completes DNA synthesis on the leading strand, whereas pol δ elongates each lagging strand fragment. Pol β is involved with DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity to	Biochemistry_Lippinco. the lagging strand needs many (discontinuous synthesis involving Okazaki fragments). In Escherichia coli (E. coli), DNA chain elongation is catalyzed by DNA pol III, using 5′-deoxyribonucleoside triphosphates as substrates. The enzyme proofreads the newly synthesized DNA, removing terminal mismatched nucleotides with its 3′→5′ exonuclease activity. RNA primers are removed by DNA pol I, using its 5′→3′ exonuclease activity. This enzyme fills the gaps with DNA, proofreading as it synthesizes. The final phosphodiester linkage is catalyzed by DNA ligase. There are at least five high-fidelity eukaryotic DNA pols. Pol α is a multisubunit enzyme, one subunit of which is a primase. Pol α 5′→3′ polymerase activity adds a short piece of DNA to the RNA primer. Pol ε completes DNA synthesis on the leading strand, whereas pol δ elongates each lagging strand fragment. Pol β is involved with DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity to
Biochemistry_Lippincott_1496	Biochemistry_Lippinco	the leading strand, whereas pol δ elongates each lagging strand fragment. Pol β is involved with DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity to proofread. Nucleoside analogs containing modified sugars can be used to block DNA chain growth. They are useful in anticancer and antiviral chemotherapy. Telomeres are stretches of highly repetitive DNA complexed with protein that protect the ends of linear chromosomes. As most cells divide and age, these sequences are shortened, contributing to senescence. In cells that do not senesce (for example, germline and cancer cells), the ribonucleoprotein telomerase employs its protein component reverse transcriptase to extend the telomeres, using its RNA component as a template. There are five classes of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting	Biochemistry_Lippinco. the leading strand, whereas pol δ elongates each lagging strand fragment. Pol β is involved with DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity to proofread. Nucleoside analogs containing modified sugars can be used to block DNA chain growth. They are useful in anticancer and antiviral chemotherapy. Telomeres are stretches of highly repetitive DNA complexed with protein that protect the ends of linear chromosomes. As most cells divide and age, these sequences are shortened, contributing to senescence. In cells that do not senesce (for example, germline and cancer cells), the ribonucleoprotein telomerase employs its protein component reverse transcriptase to extend the telomeres, using its RNA component as a template. There are five classes of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting
Biochemistry_Lippincott_1497	Biochemistry_Lippinco	of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting the nucleosomes, called linker DNA, is bound to H1. Nucleosomes can be packed more tightly to form a nucleofilament. Additional levels of organization create a chromosome. Most DNA damage can be corrected by excision repair involving recognition and removal of the damage by repair proteins, followed by replacement by DNA pols and joining by ligase. Ultraviolet radiation can cause thymine dimers that are recognized and removed in E. coli by uvrABC proteins of nucleotide excision repair. Defects in the XP proteins needed for nucleotide excision repair of thymine dimers in humans result in xeroderma pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes.	Biochemistry_Lippinco. of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting the nucleosomes, called linker DNA, is bound to H1. Nucleosomes can be packed more tightly to form a nucleofilament. Additional levels of organization create a chromosome. Most DNA damage can be corrected by excision repair involving recognition and removal of the damage by repair proteins, followed by replacement by DNA pols and joining by ligase. Ultraviolet radiation can cause thymine dimers that are recognized and removed in E. coli by uvrABC proteins of nucleotide excision repair. Defects in the XP proteins needed for nucleotide excision repair of thymine dimers in humans result in xeroderma pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes.
Biochemistry_Lippincott_1498	Biochemistry_Lippinco	pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes. Defective mismatch repair by homologous proteins in humans is associated with hereditary nonpolyposis colorectal cancer. Abnormal bases (such as uracil) are removed by DNA N-glycosylases in base excision repair, and the sugar phosphate at the apyrimidinic or apurinic site is cut out. Double-strand breaks in DNA are repaired by nonhomologous end joining (error prone) and template-requiring homologous recombination (“error-free”).	Biochemistry_Lippinco. pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes. Defective mismatch repair by homologous proteins in humans is associated with hereditary nonpolyposis colorectal cancer. Abnormal bases (such as uracil) are removed by DNA N-glycosylases in base excision repair, and the sugar phosphate at the apyrimidinic or apurinic site is cut out. Double-strand breaks in DNA are repaired by nonhomologous end joining (error prone) and template-requiring homologous recombination (“error-free”).
Biochemistry_Lippincott_1499	Biochemistry_Lippinco	Choose the ONE best answer. 0.1. A 10-year-old girl is brought by her parents to the dermatologist. She has many freckles on her face, neck, arms, and hands, and the parents report that she is unusually sensitive to sunlight. Two basal cell carcinomas are identified on her face. Based on the clinical picture, which of the following processes is most likely to be defective in this patient? A. Repair of double-strand breaks by error-prone homologous recombination B. Removal of mismatched bases from the 3′-end of Okazaki fragments by a methyl-directed process C. Removal of pyrimidine dimers from DNA by nucleotide excision repair D. Removal of uracil from DNA by base excision repair	Biochemistry_Lippinco. Choose the ONE best answer. 0.1. A 10-year-old girl is brought by her parents to the dermatologist. She has many freckles on her face, neck, arms, and hands, and the parents report that she is unusually sensitive to sunlight. Two basal cell carcinomas are identified on her face. Based on the clinical picture, which of the following processes is most likely to be defective in this patient? A. Repair of double-strand breaks by error-prone homologous recombination B. Removal of mismatched bases from the 3′-end of Okazaki fragments by a methyl-directed process C. Removal of pyrimidine dimers from DNA by nucleotide excision repair D. Removal of uracil from DNA by base excision repair
Biochemistry_Lippincott_1500	Biochemistry_Lippinco	C. Removal of pyrimidine dimers from DNA by nucleotide excision repair D. Removal of uracil from DNA by base excision repair Correct answer = C. The sensitivity to sunlight, extensive freckling on parts of the body exposed to the sun, and presence of skin cancer at a young age indicate that the patient most likely suffers from xeroderma pigmentosum (XP). These patients are deficient in any one of several XP proteins required for nucleotide excision repair of pyrimidine dimers in ultraviolet radiation– damaged DNA. Double-strand breaks are repaired by nonhomologous end joining (error prone) or homologous recombination (“error free”). Methylation is not used for strand discrimination in eukaryotic mismatch repair. Uracil is removed from DNA molecules by a specific glycosylase in base excision repair, but a defect in this process does not cause XP.	Biochemistry_Lippinco. C. Removal of pyrimidine dimers from DNA by nucleotide excision repair D. Removal of uracil from DNA by base excision repair Correct answer = C. The sensitivity to sunlight, extensive freckling on parts of the body exposed to the sun, and presence of skin cancer at a young age indicate that the patient most likely suffers from xeroderma pigmentosum (XP). These patients are deficient in any one of several XP proteins required for nucleotide excision repair of pyrimidine dimers in ultraviolet radiation– damaged DNA. Double-strand breaks are repaired by nonhomologous end joining (error prone) or homologous recombination (“error free”). Methylation is not used for strand discrimination in eukaryotic mismatch repair. Uracil is removed from DNA molecules by a specific glycosylase in base excision repair, but a defect in this process does not cause XP.
Biochemistry_Lippincott_1501	Biochemistry_Lippinco	0.2. Telomeres are complexes of DNA and protein that protect the ends of linear chromosomes. In most normal human somatic cells, telomeres shorten with each division. In stem cells and in cancer cells, however, telomeric length is maintained. In the synthesis of telomeres: A. telomerase, a ribonucleoprotein, provides both the RNA and the protein needed for synthesis. B. the RNA of telomerase serves as a primer. C. the RNA of telomerase is a ribozyme. D. the protein of telomerase is a DNA-directed DNA polymerase. E. the shorter 3′→5′ strand gets extended. F. the direction of synthesis is 3′→5′.	Biochemistry_Lippinco. 0.2. Telomeres are complexes of DNA and protein that protect the ends of linear chromosomes. In most normal human somatic cells, telomeres shorten with each division. In stem cells and in cancer cells, however, telomeric length is maintained. In the synthesis of telomeres: A. telomerase, a ribonucleoprotein, provides both the RNA and the protein needed for synthesis. B. the RNA of telomerase serves as a primer. C. the RNA of telomerase is a ribozyme. D. the protein of telomerase is a DNA-directed DNA polymerase. E. the shorter 3′→5′ strand gets extended. F. the direction of synthesis is 3′→5′.
Biochemistry_Lippincott_1502	Biochemistry_Lippinco	C. the RNA of telomerase is a ribozyme. D. the protein of telomerase is a DNA-directed DNA polymerase. E. the shorter 3′→5′ strand gets extended. F. the direction of synthesis is 3′→5′. Correct answer = A. Telomerase is a ribonucleoprotein particle required for telomere maintenance. Telomerase contains an RNA that serves as the template, not the primer, for the synthesis of telomeric DNA by the reverse transcriptase of telomerase. Telomeric RNA has no catalytic activity. As a reverse transcriptase, telomerase synthesizes DNA using its RNA template and so is an RNA-directed DNA polymerase. The direction of synthesis, as with all DNA synthesis, is 5′→3′, and it is the 3′-end of the already longer 5′→3′ strand that gets extended.	Biochemistry_Lippinco. C. the RNA of telomerase is a ribozyme. D. the protein of telomerase is a DNA-directed DNA polymerase. E. the shorter 3′→5′ strand gets extended. F. the direction of synthesis is 3′→5′. Correct answer = A. Telomerase is a ribonucleoprotein particle required for telomere maintenance. Telomerase contains an RNA that serves as the template, not the primer, for the synthesis of telomeric DNA by the reverse transcriptase of telomerase. Telomeric RNA has no catalytic activity. As a reverse transcriptase, telomerase synthesizes DNA using its RNA template and so is an RNA-directed DNA polymerase. The direction of synthesis, as with all DNA synthesis, is 5′→3′, and it is the 3′-end of the already longer 5′→3′ strand that gets extended.
Biochemistry_Lippincott_1503	Biochemistry_Lippinco	0.3. While studying the structure of a small gene that was sequenced during the Human Genome Project, an investigator notices that one strand of the DNA molecule contains 20 A, 25 G, 30 C, and 22 T. How many of each base is found in the complete double-stranded molecule? A. A=40,G =50,C =60,T =44 E. A=42,G =55,C =55,T =42 B. A=44,G =60,C =50,T =40 C. A=45,G =45,C =52,T =52 D. A=50,G =47,C =50,T =47 Correct answer = B. The two DNA strands are complementary to each other, with A base-paired with T and G base-paired with C. So, for example, the 20 A on the first strand would be paired with 20 T on the second strand, the 25 G on the first strand would be paired with 25 C on the second strand, and so forth. When these are all added together, the correct numbers of each base are indicated in choice B. Notice that, in the correct answer, A = T and G = C. 0.4. List the order in which the following enzymes participate in prokaryotic replication. A. Ligase	Biochemistry_Lippinco. 0.3. While studying the structure of a small gene that was sequenced during the Human Genome Project, an investigator notices that one strand of the DNA molecule contains 20 A, 25 G, 30 C, and 22 T. How many of each base is found in the complete double-stranded molecule? A. A=40,G =50,C =60,T =44 E. A=42,G =55,C =55,T =42 B. A=44,G =60,C =50,T =40 C. A=45,G =45,C =52,T =52 D. A=50,G =47,C =50,T =47 Correct answer = B. The two DNA strands are complementary to each other, with A base-paired with T and G base-paired with C. So, for example, the 20 A on the first strand would be paired with 20 T on the second strand, the 25 G on the first strand would be paired with 25 C on the second strand, and so forth. When these are all added together, the correct numbers of each base are indicated in choice B. Notice that, in the correct answer, A = T and G = C. 0.4. List the order in which the following enzymes participate in prokaryotic replication. A. Ligase
Biochemistry_Lippincott_1504	Biochemistry_Lippinco	0.4. List the order in which the following enzymes participate in prokaryotic replication. A. Ligase B. Polymerase I (3′→5′ exonuclease activity) C. Polymerase I (5′→3′ exonuclease activity) D. Polymerase I (5′→3′ polymerase activity) E. Polymerase III F. Primase Correct answer: F, E, C, D, B, A. Primase makes the RNA primer; polymerase (pol) III extends the primer with DNA (and proofreads); pol I removes the primer with its 5′→3′ exonuclease activity, fills in the gap with its 5′→3′ polymerase activity, and removes errors with its 3′→5′ exonuclease activity; and ligase makes the 5′→3′-phosphodiester bond that links the DNA made by pols I and III. 0.5. Dideoxynucleotides lack a 3′-hydroxyl group. Why would incorporation of a dideoxynucleotide into DNA stop replication? The lack of the 3′-OH group prevents formation of the 3′-hydroxyl → 5′phosphate bond that links one nucleotide to the next in DNA. RNA Structure, Synthesis, and Processing 31	Biochemistry_Lippinco. 0.4. List the order in which the following enzymes participate in prokaryotic replication. A. Ligase B. Polymerase I (3′→5′ exonuclease activity) C. Polymerase I (5′→3′ exonuclease activity) D. Polymerase I (5′→3′ polymerase activity) E. Polymerase III F. Primase Correct answer: F, E, C, D, B, A. Primase makes the RNA primer; polymerase (pol) III extends the primer with DNA (and proofreads); pol I removes the primer with its 5′→3′ exonuclease activity, fills in the gap with its 5′→3′ polymerase activity, and removes errors with its 3′→5′ exonuclease activity; and ligase makes the 5′→3′-phosphodiester bond that links the DNA made by pols I and III. 0.5. Dideoxynucleotides lack a 3′-hydroxyl group. Why would incorporation of a dideoxynucleotide into DNA stop replication? The lack of the 3′-OH group prevents formation of the 3′-hydroxyl → 5′phosphate bond that links one nucleotide to the next in DNA. RNA Structure, Synthesis, and Processing 31
Biochemistry_Lippincott_1505	Biochemistry_Lippinco	The lack of the 3′-OH group prevents formation of the 3′-hydroxyl → 5′phosphate bond that links one nucleotide to the next in DNA. RNA Structure, Synthesis, and Processing 31 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW	Biochemistry_Lippinco. The lack of the 3′-OH group prevents formation of the 3′-hydroxyl → 5′phosphate bond that links one nucleotide to the next in DNA. RNA Structure, Synthesis, and Processing 31 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW
Biochemistry_Lippincott_1506	Biochemistry_Lippinco	The genetic master plan of an organism is contained in the sequence of deoxyribonucleotides in its DNA. However, it is through ribonucleic acid (RNA), the “working copies” of DNA, that the master plan is expressed (Fig. 31.1). The copying process, during which a DNA strand serves as a template for the synthesis of RNA, is called transcription. Transcription produces messenger RNA (mRNA), which are translated into sequences of amino acids (proteins), and ribosomal RNA (rRNA), transfer RNA (tRNA), and additional RNA molecules that perform specialized structural, catalytic, and regulatory functions and are not translated. That is, they are noncoding RNA (ncRNA). Therefore, the final product of gene expression can be RNA or protein, depending upon the gene. [Note: Only ~2% of the genome encodes proteins.] A central feature of transcription is that it is highly selective. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made.	Biochemistry_Lippinco. The genetic master plan of an organism is contained in the sequence of deoxyribonucleotides in its DNA. However, it is through ribonucleic acid (RNA), the “working copies” of DNA, that the master plan is expressed (Fig. 31.1). The copying process, during which a DNA strand serves as a template for the synthesis of RNA, is called transcription. Transcription produces messenger RNA (mRNA), which are translated into sequences of amino acids (proteins), and ribosomal RNA (rRNA), transfer RNA (tRNA), and additional RNA molecules that perform specialized structural, catalytic, and regulatory functions and are not translated. That is, they are noncoding RNA (ncRNA). Therefore, the final product of gene expression can be RNA or protein, depending upon the gene. [Note: Only ~2% of the genome encodes proteins.] A central feature of transcription is that it is highly selective. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made.
Biochemistry_Lippincott_1507	Biochemistry_Lippinco	proteins.] A central feature of transcription is that it is highly selective. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made. This selectivity is due, at least in part, to signals embedded in the nucleotide sequence of the DNA. These signals instruct the RNA polymerase where to start, how often to start, and where to stop transcription. Several regulatory proteins are also involved in this selection process. The biochemical differentiation of an organism’s tissues is ultimately a result of the selectivity of the transcription process. [Note: This selectivity of transcription is in contrast to the “all-or-none” nature of genomic replication.] Another important feature of transcription is that many RNA transcripts that initially are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the	Biochemistry_Lippinco. proteins.] A central feature of transcription is that it is highly selective. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made. This selectivity is due, at least in part, to signals embedded in the nucleotide sequence of the DNA. These signals instruct the RNA polymerase where to start, how often to start, and where to stop transcription. Several regulatory proteins are also involved in this selection process. The biochemical differentiation of an organism’s tissues is ultimately a result of the selectivity of the transcription process. [Note: This selectivity of transcription is in contrast to the “all-or-none” nature of genomic replication.] Another important feature of transcription is that many RNA transcripts that initially are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the
Biochemistry_Lippincott_1508	Biochemistry_Lippinco	are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the inactive primary transcript into a functional molecule. The transcriptome is the complete set of RNA transcripts expressed by a genome.	Biochemistry_Lippinco. are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the inactive primary transcript into a functional molecule. The transcriptome is the complete set of RNA transcripts expressed by a genome.
Biochemistry_Lippincott_1509	Biochemistry_Lippinco	II. RNA STRUCTURE There are three major types of RNA that participate in the process of protein synthesis: rRNA, tRNA, and mRNA. Like DNA, these RNA are unbranched polymeric molecules composed of nucleoside monophosphates joined together by 3′→5′-phosphodiester bonds (see p. 412). However, they differ from DNA in several ways. For example, they are considerably smaller than DNA, contain ribose instead of deoxyribose and uracil instead of thymine, and exist as single strands that are capable of folding into complex structures. The three major types of RNA also differ from each other in size, function, and special structural modifications. [Note: In eukaryotes, additional small ncRNA molecules found in the nucleolus (snoRNA), nucleus (snRNA), and cytoplasm (microRNA [miRNA]) perform specialized functions as described on pp. 441, 442, and 475.]	Biochemistry_Lippinco. II. RNA STRUCTURE There are three major types of RNA that participate in the process of protein synthesis: rRNA, tRNA, and mRNA. Like DNA, these RNA are unbranched polymeric molecules composed of nucleoside monophosphates joined together by 3′→5′-phosphodiester bonds (see p. 412). However, they differ from DNA in several ways. For example, they are considerably smaller than DNA, contain ribose instead of deoxyribose and uracil instead of thymine, and exist as single strands that are capable of folding into complex structures. The three major types of RNA also differ from each other in size, function, and special structural modifications. [Note: In eukaryotes, additional small ncRNA molecules found in the nucleolus (snoRNA), nucleus (snRNA), and cytoplasm (microRNA [miRNA]) perform specialized functions as described on pp. 441, 442, and 475.]
Biochemistry_Lippincott_1510	Biochemistry_Lippinco	A. Ribosomal RNA rRNA are found in association with several proteins as components of the ribosomes, the complex structures that serve as the sites for protein synthesis (see p. 451). Prokaryotic cells contain three distinct size species of rRNA (23S, 16S, and 5S, where S is the Svedberg unit for sedimentation rate that is determined by the size and shape of the particle), as shown in Figure 31.2. Eukaryotic cells contain four rRNA species (28S, 18S, 5.8S, and 5S). Together, rRNA make up ~80% of the total RNA in the cell. [Note: Some RNA function as catalysts, for example, an rRNA in protein synthesis (see p. 455). RNA with catalytic activity is termed a ribozyme.]	Biochemistry_Lippinco. A. Ribosomal RNA rRNA are found in association with several proteins as components of the ribosomes, the complex structures that serve as the sites for protein synthesis (see p. 451). Prokaryotic cells contain three distinct size species of rRNA (23S, 16S, and 5S, where S is the Svedberg unit for sedimentation rate that is determined by the size and shape of the particle), as shown in Figure 31.2. Eukaryotic cells contain four rRNA species (28S, 18S, 5.8S, and 5S). Together, rRNA make up ~80% of the total RNA in the cell. [Note: Some RNA function as catalysts, for example, an rRNA in protein synthesis (see p. 455). RNA with catalytic activity is termed a ribozyme.]
Biochemistry_Lippincott_1511	Biochemistry_Lippinco	B. Transfer RNA tRNA are the smallest (4S) of the three major types of RNA molecules. There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins. Together, tRNA make up ~15% of the total RNA in the cell. The tRNA molecules contain a high percentage of unusual (modified) bases, for example, dihydrouracil (see Fig. 22.2, p. 292), and have extensive intrachain base-pairing (Fig. 31.3) that leads to characteristic secondary and tertiary structure. Each tRNA serves as an adaptor molecule that carries its specific amino acid, covalently attached to its 3′-end, to the site of protein synthesis. There, it recognizes the genetic code sequence on an mRNA, which specifies the addition of that amino acid to the growing peptide chain (see p. 447).	Biochemistry_Lippinco. B. Transfer RNA tRNA are the smallest (4S) of the three major types of RNA molecules. There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins. Together, tRNA make up ~15% of the total RNA in the cell. The tRNA molecules contain a high percentage of unusual (modified) bases, for example, dihydrouracil (see Fig. 22.2, p. 292), and have extensive intrachain base-pairing (Fig. 31.3) that leads to characteristic secondary and tertiary structure. Each tRNA serves as an adaptor molecule that carries its specific amino acid, covalently attached to its 3′-end, to the site of protein synthesis. There, it recognizes the genetic code sequence on an mRNA, which specifies the addition of that amino acid to the growing peptide chain (see p. 447).
Biochemistry_Lippincott_1512	Biochemistry_Lippinco	C. Messenger RNA mRNA comprises only ~5% of the RNA in a cell, yet is by far the most heterogeneous type of RNA in size and base sequence. mRNA is coding RNA in that it carries genetic information from DNA for use in protein synthesis. In eukaryotes, this involves transport of mRNA out of the nucleus and into the cytosol. An mRNA carrying information from more than one gene is polycistronic (cistron = gene). Polycistronic mRNA is characteristic of prokaryotes. An mRNA carrying information from only one gene is monocistronic and is characteristic of eukaryotes. In addition to the protein-coding regions that can be translated, mRNA contains untranslated regions at its 5′-and 3′-ends (Fig. 31.4). Special structural characteristics of eukaryotic (but not prokaryotic) mRNA include a long sequence of adenine nucleotides (a poly-A tail) on the 3′-end of the RNA, plus a cap on the 5′-end consisting of a molecule of 7-methylguanosine attached through an unusual (5′→5′) triphosphate linkage.	Biochemistry_Lippinco. C. Messenger RNA mRNA comprises only ~5% of the RNA in a cell, yet is by far the most heterogeneous type of RNA in size and base sequence. mRNA is coding RNA in that it carries genetic information from DNA for use in protein synthesis. In eukaryotes, this involves transport of mRNA out of the nucleus and into the cytosol. An mRNA carrying information from more than one gene is polycistronic (cistron = gene). Polycistronic mRNA is characteristic of prokaryotes. An mRNA carrying information from only one gene is monocistronic and is characteristic of eukaryotes. In addition to the protein-coding regions that can be translated, mRNA contains untranslated regions at its 5′-and 3′-ends (Fig. 31.4). Special structural characteristics of eukaryotic (but not prokaryotic) mRNA include a long sequence of adenine nucleotides (a poly-A tail) on the 3′-end of the RNA, plus a cap on the 5′-end consisting of a molecule of 7-methylguanosine attached through an unusual (5′→5′) triphosphate linkage.
Biochemistry_Lippincott_1513	Biochemistry_Lippinco	of adenine nucleotides (a poly-A tail) on the 3′-end of the RNA, plus a cap on the 5′-end consisting of a molecule of 7-methylguanosine attached through an unusual (5′→5′) triphosphate linkage. The mechanisms for modifying mRNA to create these special structural characteristics are discussed on pp. 441–442.	Biochemistry_Lippinco. of adenine nucleotides (a poly-A tail) on the 3′-end of the RNA, plus a cap on the 5′-end consisting of a molecule of 7-methylguanosine attached through an unusual (5′→5′) triphosphate linkage. The mechanisms for modifying mRNA to create these special structural characteristics are discussed on pp. 441–442.
Biochemistry_Lippincott_1514	Biochemistry_Lippinco	III. PROKARYOTIC GENE TRANSCRIPTION The structure of magnesium-requiring RNA polymerase (RNA pol), the signals that control transcription, and the varieties of modification that RNA transcripts can undergo differ among organisms, particularly from prokaryotes to eukaryotes. Therefore, the discussions of prokaryotic and eukaryotic transcription are presented separately. A. Prokaryotic RNA polymerase	Biochemistry_Lippinco. III. PROKARYOTIC GENE TRANSCRIPTION The structure of magnesium-requiring RNA polymerase (RNA pol), the signals that control transcription, and the varieties of modification that RNA transcripts can undergo differ among organisms, particularly from prokaryotes to eukaryotes. Therefore, the discussions of prokaryotic and eukaryotic transcription are presented separately. A. Prokaryotic RNA polymerase
Biochemistry_Lippincott_1515	Biochemistry_Lippinco	In bacteria, one species of RNA pol synthesizes all of the RNA except for the short RNA primers needed for DNA replication [Note: RNA primers are synthesized by the specialized, monomeric enzyme primase (see p. 418).] RNA pol is a multisubunit enzyme that recognizes a nucleotide sequence (the promoter region) at the beginning of a length of DNA that is to be transcribed. It next makes a complementary RNA copy of the DNA template strand and then recognizes the end of the DNA sequence to be transcribed (the termination region). RNA is synthesized from its 5′-end to its 3′-end, antiparallel to its DNA template strand (see p. 415). The template is copied as it is in DNA synthesis, in which a guanine (G) on the DNA specifies a cytosine (C) in the RNA, a C specifies a G, a thymine (T) specifies an adenine (A), but an A specifies a uracil (U) instead of a T (Fig. 31.5). The RNA, then, is complementary to the DNA template (antisense, minus) strand and identical to the coding (sense, plus)	Biochemistry_Lippinco. In bacteria, one species of RNA pol synthesizes all of the RNA except for the short RNA primers needed for DNA replication [Note: RNA primers are synthesized by the specialized, monomeric enzyme primase (see p. 418).] RNA pol is a multisubunit enzyme that recognizes a nucleotide sequence (the promoter region) at the beginning of a length of DNA that is to be transcribed. It next makes a complementary RNA copy of the DNA template strand and then recognizes the end of the DNA sequence to be transcribed (the termination region). RNA is synthesized from its 5′-end to its 3′-end, antiparallel to its DNA template strand (see p. 415). The template is copied as it is in DNA synthesis, in which a guanine (G) on the DNA specifies a cytosine (C) in the RNA, a C specifies a G, a thymine (T) specifies an adenine (A), but an A specifies a uracil (U) instead of a T (Fig. 31.5). The RNA, then, is complementary to the DNA template (antisense, minus) strand and identical to the coding (sense, plus)
Biochemistry_Lippincott_1516	Biochemistry_Lippinco	an adenine (A), but an A specifies a uracil (U) instead of a T (Fig. 31.5). The RNA, then, is complementary to the DNA template (antisense, minus) strand and identical to the coding (sense, plus) strand, with U replacing T. Within the DNA molecule, regions of both strands can serve as templates for transcription. For a given gene, however, only one of the two DNA strands can be the template. Which strand is used is determined by the location of the promoter for that gene. Transcription by RNA pol involves a core enzyme and several auxiliary proteins.	Biochemistry_Lippinco. an adenine (A), but an A specifies a uracil (U) instead of a T (Fig. 31.5). The RNA, then, is complementary to the DNA template (antisense, minus) strand and identical to the coding (sense, plus) strand, with U replacing T. Within the DNA molecule, regions of both strands can serve as templates for transcription. For a given gene, however, only one of the two DNA strands can be the template. Which strand is used is determined by the location of the promoter for that gene. Transcription by RNA pol involves a core enzyme and several auxiliary proteins.
Biochemistry_Lippincott_1517	Biochemistry_Lippinco	1. Core enzyme: Five of the enzyme’s peptide subunits, 2 α, 1 β, 1 β′, and 1 Ω, are required for enzyme assembly (α, Ω), template binding (β′), and the 5′→3′ polymerase activity (β) and together are referred to as the core enzyme (Fig. 31.6). However, this enzyme lacks specificity (that is, it cannot recognize the promoter region on the DNA template). 2. Holoenzyme: The σ subunit (sigma factor) enables RNA pol to recognize promoter regions on the DNA. The σ subunit plus the core enzyme make up the holoenzyme. [Note: Different σ factors recognize different groups of genes, with σ70 predominating.] B. Steps in RNA synthesis The process of transcription of a typical gene of Escherichia coli (E. coli) can be divided into three phases: initiation, elongation, and termination. A transcription unit extends from the promoter to the termination region, and the initial product of transcription by RNA pol is termed the primary transcript.	Biochemistry_Lippinco. 1. Core enzyme: Five of the enzyme’s peptide subunits, 2 α, 1 β, 1 β′, and 1 Ω, are required for enzyme assembly (α, Ω), template binding (β′), and the 5′→3′ polymerase activity (β) and together are referred to as the core enzyme (Fig. 31.6). However, this enzyme lacks specificity (that is, it cannot recognize the promoter region on the DNA template). 2. Holoenzyme: The σ subunit (sigma factor) enables RNA pol to recognize promoter regions on the DNA. The σ subunit plus the core enzyme make up the holoenzyme. [Note: Different σ factors recognize different groups of genes, with σ70 predominating.] B. Steps in RNA synthesis The process of transcription of a typical gene of Escherichia coli (E. coli) can be divided into three phases: initiation, elongation, and termination. A transcription unit extends from the promoter to the termination region, and the initial product of transcription by RNA pol is termed the primary transcript.
Biochemistry_Lippincott_1518	Biochemistry_Lippinco	1. Initiation: Transcription begins with the binding of the RNA pol holoenzyme to a region of the DNA known as the promoter, which is not transcribed. The prokaryotic promoter contains characteristic consensus sequences (Fig. 31.7). [Note: Consensus sequences are idealized sequences in which the base shown at each position is the base most frequently (but not necessarily always) encountered at that position.] Those that are recognized by prokaryotic RNA pol σ factors include the following. a.	Biochemistry_Lippinco. 1. Initiation: Transcription begins with the binding of the RNA pol holoenzyme to a region of the DNA known as the promoter, which is not transcribed. The prokaryotic promoter contains characteristic consensus sequences (Fig. 31.7). [Note: Consensus sequences are idealized sequences in which the base shown at each position is the base most frequently (but not necessarily always) encountered at that position.] Those that are recognized by prokaryotic RNA pol σ factors include the following. a.
Biochemistry_Lippincott_1519	Biochemistry_Lippinco	a. –35 Sequence: A consensus sequence (5′-TTGACA-3′), centered about 35 bases to the left of the transcription start site (see Fig. 31.7), is the initial point of contact for the holoenzyme, and a closed complex is formed. [Note: By convention, the regulatory sequences that control transcription are designated by the 5′→3′ nucleotide sequence on the coding strand. A base in the promoter region is assigned a negative number if it occurs prior to (to the left of, toward the 5′-end of, or “upstream” of) the transcription start site. Therefore, the TTGACA sequence is centered at approximately base −35. The first base at the transcription start site is assigned a position of +1. There is no base designated “0”.] b.	Biochemistry_Lippinco. a. –35 Sequence: A consensus sequence (5′-TTGACA-3′), centered about 35 bases to the left of the transcription start site (see Fig. 31.7), is the initial point of contact for the holoenzyme, and a closed complex is formed. [Note: By convention, the regulatory sequences that control transcription are designated by the 5′→3′ nucleotide sequence on the coding strand. A base in the promoter region is assigned a negative number if it occurs prior to (to the left of, toward the 5′-end of, or “upstream” of) the transcription start site. Therefore, the TTGACA sequence is centered at approximately base −35. The first base at the transcription start site is assigned a position of +1. There is no base designated “0”.] b.
Biochemistry_Lippincott_1520	Biochemistry_Lippinco	Pribnow box: The holoenzyme moves and covers a second consensus sequence (5′-TATAAT-3′), centered at about −10 (see Fig. 31.7), which is the site of melting (unwinding) of a short stretch (~14 base pairs) of DNA. This initial melting converts the closed initiation complex to an open complex known as a transcription bubble. [Note: A mutation in either the −10 or the −35 sequence can affect the transcription of the gene controlled by the mutant promoter.] 2.	Biochemistry_Lippinco. Pribnow box: The holoenzyme moves and covers a second consensus sequence (5′-TATAAT-3′), centered at about −10 (see Fig. 31.7), which is the site of melting (unwinding) of a short stretch (~14 base pairs) of DNA. This initial melting converts the closed initiation complex to an open complex known as a transcription bubble. [Note: A mutation in either the −10 or the −35 sequence can affect the transcription of the gene controlled by the mutant promoter.] 2.
Biochemistry_Lippincott_1521	Biochemistry_Lippinco	Elongation: Once the promoter has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues (Fig. 31.8), mediated by the polymerase. [Note: Unwinding generates supercoils in the DNA that can be relieved by DNA topoisomerases (see p. 417).] RNA pol begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase begins when the transcript (typically starting with a purine) exceeds 10 nucleotides in length. Sigma is then released, and the core enzyme is able to leave (clear) the promoter and move along the template strand in a processive manner, serving as its own sliding clamp. During transcription, a short DNA–RNA hybrid helix is formed (see Fig. 31.8). Like DNA pol, RNA pol uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In	Biochemistry_Lippinco. Elongation: Once the promoter has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues (Fig. 31.8), mediated by the polymerase. [Note: Unwinding generates supercoils in the DNA that can be relieved by DNA topoisomerases (see p. 417).] RNA pol begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase begins when the transcript (typically starting with a purine) exceeds 10 nucleotides in length. Sigma is then released, and the core enzyme is able to leave (clear) the promoter and move along the template strand in a processive manner, serving as its own sliding clamp. During transcription, a short DNA–RNA hybrid helix is formed (see Fig. 31.8). Like DNA pol, RNA pol uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In
Biochemistry_Lippincott_1522	Biochemistry_Lippinco	triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In contrast to DNA pol, RNA pol does not require a primer and does not have a 3′→5′ exonuclease domain for proofreading. [Note: Misincorporation of a ribonucleotide causes RNA pol to pause, backtrack, cleave the transcript, and restart. Nonetheless, transcription has a higher error rate than does replication.] 3.	Biochemistry_Lippinco. triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In contrast to DNA pol, RNA pol does not require a primer and does not have a 3′→5′ exonuclease domain for proofreading. [Note: Misincorporation of a ribonucleotide causes RNA pol to pause, backtrack, cleave the transcript, and restart. Nonetheless, transcription has a higher error rate than does replication.] 3.