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Chapter 26 Nucleosides, Nucleotides, and Nucleic Acids 26.1 Pyrimidines and Purines Pyrimidines and Purines In order to understand the structure and properties of DNA and RNA, we need to look at their structural components. We begin with certain heterocyclic aromatic compounds called pyrimidines and purines. Pyrimidines and Purines Pyrimidine and purine are the names of the parent compounds of two types of nitrogencontaining heterocyclic aromatic compounds. 6 7 N1 5 4 N 2 3 Pyrimidine N 5 N H 4 6 N1 8 9 N 3 Purine 2 Pyrimidines and Purines Amino-substituted derivatives of pyrimidine and purine have the structures expected from their names. H H NH2 N N N H H2N N H N N H H 4-Aminopyrimidine 6-Aminopurine Pyrimidines and Purines But hydroxy-substituted pyrimidines and purines exist in keto, rather than enol, forms. H H HO H H N N H O N N H enol keto H Pyrimidines and Purines But hydroxy-substituted pyrimidines and purines exist in keto, rather than enol, forms. OH N N N H N O N H H N H H enol N keto N H H Important Pyrimidines Pyrimidines that occur in DNA are cytosine and thymine. Cytosine and uracil are the pyrimidines in RNA. O O NH2 CH3 HN HN HN O N H Uracil O N H Thymine O N H Cytosine Important Purines Adenine and guanine are the principal purines of both DNA and RNA. NH2 O N N N N H Adenine N HN H2N N N H Guanine Caffeine and Theobromine Caffeine (coffee) and theobromine (coffee and tea) are naturally occurring purines. O H3C O N N N O CH3 N CH3 Caffeine N HN O CH3 N N CH3 Theobromine 26.2 Nucleosides Nucleosides The classical structural definition is that a nucleoside is a pyrimidine or purine N-glycoside of D-ribofuranose or 2-deoxy-D-ribofuranose. Informal use has extended this definition to apply to purine or pyrimidine N-glycosides of almost any carbohydrate. The purine or pyrimidine part of a nucleoside is referred to as a purine or pyrimidine base. Table 26.2 Pyrimidine nucleosides NH2 N N Cytidine HOCH2 HO O O OH Cytidine occurs in RNA; its 2-deoxy analog occurs in DNA. Table 26.2 Pyrimidine nucleosides O H3C Thymidine HOCH2 NH N O O HO Thymidine occurs in DNA. Table 26.2 Pyrimidine nucleosides O NH N Uridine HOCH2 HO O O OH Uridine occurs in RNA. Table 26.2 Purine nucleosides NH2 N Adenosine HOCH2 O HO N N N OH Adenosine occurs in RNA; its 2-deoxy analog occurs in DNA. Table 26.2 Purine nucleosides O N Guanosine HOCH2 O HO N NH N NH2 OH Guanosine occurs in RNA; its 2-deoxy analog occurs in DNA. 26.3 Nucleotides Nucleotides are phosphoric acid esters of nucleosides. Adenosine 5'-Monophosphate (AMP) Adenosine 5'-monophosphate (AMP) is also called 5'-adenylic acid. NH2 N N O 5' HO P OCH2 O HO N 1' 4' 3' HO 2' OH N Adenosine Diphosphate (ADP) NH2 N O HO P O HO N O P OCH2 O N HO HO OH N Adenosine Triphosphate (ATP) ATP is an important molecule in several biochemical processes including: energy storage (Sections 26.4-26.5) phosphorylation NH2 N O HO O P O P O HO HO N O P OCH2 O N HO HO OH N ATP and Phosphorylation HOCH2 ATP + HO HO O HO This is the first step in the metabolism of glucose. OH hexokinase O ADP + (HO)2POCH2 O HO HO HO OH cAMP and cGMP Cyclic AMP and cyclic GMP are "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP. NH2 N CH2 O O O HO N N N P O OH Cyclic adenosine monophosphate (cAMP) cAMP and cGMP Cyclic AMP and cyclic GMP are "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP. O O HO O N CH2 O N NH N NH2 P O OH Cyclic guanosine monophosphate (cGMP) 26.4 Bioenergetics Bioenergetics Bioenergetics is the thermodynamics of biological processes. Emphasis is on free energy changes (DG). When DG is negative, reaction is spontaneous in the direction written. When DG is 0, reaction is at equilibrium. When DG is positive, reaction is not spontaneous in direction written. Standard Free Energy (DG°) mA(aq) nB(aq) Sign and magnitude of DG depends on what the reactants and products are and their concentrations. In order to focus on reactants and products, define a standard state. The standard concentration is 1 M (for a reaction in homogeneous solution). DG in the standard state is called the standard free-energy change and given the symbol DG°. Standard Free Energy (DG°) mA(aq) nB(aq) Exergonic: An exergonic reaction is one for which the sign of DG° is negative. Endergonic: An exergonic reaction is one for which the sign of DG° is positive. Standard Free Energy (DG°) mA(aq) nB(aq) It is useful to define a special standard state for biological reactions. This special standard state is one for which the pH = 7. The free-energy change for a process under these conditions is symbolized as DG°'. 26.5 ATP and Bioenergetics Hydrolysis of ATP ATP + H2O ADP + HPO42– DG°' for hydrolysis of ATP to ADP is –31 kJ/mol. Relative to ADP + HPO42–, ATP is a "highenergy" compound. When coupled to some other process, the conversion of ATP to ADP can provide the free energy to transform an endergonic process to an exergonic one. Glutamic Acid to Glutamine O –OCCH O – CH CHCO 2 2 + NH4+ +NH 3 DG°' = +14 kJ O Reaction is endergonic. O H2NCCH2CH2CHCO– +NH 3 + H2O Glutamic Acid to Glutamine O O –OCCH CH CHCO– 2 2 + NH4+ + ATP +NH 3 Reaction becomes exergonic when coupled to the hydrolysis of ATP. DG°' = –17 kJ O O H2NCCH2CH2CHCO– +NH 3 + HPO42– + ADP Glutamic Acid to Glutamine O O –OCCH CH CHCO– 2 2 + ATP +NH 3 Mechanism involves phosphorylation of glutamic acid. O –O P –O O O OCCH2CH2CHCO– +NH 3 + ADP Glutamic Acid to Glutamine O O H2NCCH2CH2CHCO– + HPO42– +NH 3 O –O P –O O Followed by reaction of phosphorylated glutamic acid with ammonia. O OCCH2CH2CHCO– +NH 3 + NH3 26.6 Phosphodiesters, Oligonucleotides, and Polynucleotides Phosphodiesters A phosphodiester linkage between two nucleotides is analogous to a peptide bond between two amino acids. Two nucleotides joined by a phosphodiester linkage gives a dinucleotide. Three nucleotides joined by two phosphodiester linkages gives a trinucleotide, etc. (See next slide) A polynucleotide of about 50 or fewer nucleotides is called an oligonucleotide. Fig. 26.1 The trinucleotide ATG free 5' end phosphodiester linkages between 3' of one nucleotide and 5' of the next A T G free 3' end 26.7 Nucleic Acids Nucleic acids are polynucleotides. Nucleic Acids Nucleic acids first isolated in 1869 (Johann Miescher). Oswald Avery discovered (1945) that a substance which caused a change in the genetically transmitted characteristics of a bacterium was DNA. Scientists revised their opinion of the function of DNA and began to suspect it was the major functional component of genes. Composition of DNA Erwin Chargaff (Columbia Univ.) studied DNAs from various sources and analyzed the distribution of purines and pyrimidines in them. The distribution of the bases adenine (A), guanine (G), thymine (T), and cytosine (C) varied among species. But the total purines (A and G) and the total pyrimidines (T and C) were always equal. Moreover: %A = %T, and %G = %C Composition of Human DNA For example: Purine Pyrimidine Adenine (A) 30.3% Thymine (T) 30.3% Guanine (G) 19.5% Cytosine (C) 19.9% Total purines: 49.8% Total pyrimidines: 50.1% Structure of DNA James D. Watson and Francis H. C. Crick proposed a structure for DNA in 1953. Watson and Crick's structure was based on: •Chargaff's observations •X-ray crystallographic data of Maurice Wilkins and Rosalind Franklin •Model building 26.8 Secondary Structure of DNA: The Double Helix Base Pairing Watson and Crick proposed that A and T were present in equal amounts in DNA because of complementary hydrogen bonding. 2-deoxyribose A T 2-deoxyribose Base Pairing Watson and Crick proposed that A and T were present in equal amounts in DNA because of complementary hydrogen bonding. Base Pairing Likewise, the amounts of G and C in DNA were equal because of complementary hydrogen bonding. 2-deoxyribose 2-deoxyribose G C Base Pairing Likewise, the amounts of G and C in DNA were equal because of complementary hydrogen bonding. The DNA Duplex Watson and Crick proposed a double-stranded structure for DNA in which a purine or pyrimidine base in one chain is hydrogen bonded to its complement in the other. •Gives proper Chargaff ratios (A=T and G=C) •Because each pair contains one purine and one pyrimidine, the A---T and G---C distances between strands are approximately equal. •Complementarity between strands suggests a mechanism for copying genetic information. O 3' Fig. 26.4 O C 5' O 5' G O Two antiparallel strands of DNA are paired by hydrogen bonds between purine and pyrimidine bases. O O P O 3' O 3' Ğ O P O O T 5' A O O P O O T 5' A O O P O O O G 5' O 3' O O O P O 3' O 3' Ğ Ğ O 5' O O O P O 3' O 3' Ğ Ğ O 5' O O C 5' O Ğ Fig. 26.5 Helical structure of DNA. The purine and pyrimidine bases are on the inside, sugars and phosphates on the outside. 26.9 Tertiary Structure of DNA: Supercoils DNA is coiled A strand of DNA is too long (about 3 cm in length) to fit inside a cell unless it is coiled. Random coiling would reduce accessibility to critical regions. Efficient coiling of DNA is accomplished with the aid of proteins called histones. Histones Histones are proteins rich in basic amino acids such as lysine and arginine. Histones are positively charged at biological pH. DNA is negatively charged. DNA winds around histone proteins to form nucleosomes. Histones Each nucleosome contains one and three-quarters turns of coil = 146 base pairs. Linker contains about 50 base pairs. Histones Nucleosome = Histone proteins + Supercoiled DNA 26.10 Replication of DNA Fig. 26.8 DNA Replication Fig. 26.8 DNA Replication Fig. 26.8 DNA Replication Fig. 26.8 DNA Replication Elongation of the Growing DNA Chain The free 3'-OH group of the growing DNA chain reacts with the 5'-triphosphate of the appropriate nucleotide. Fig. 26.9: Chain Elongation OH Adenine, Guanine, Cytosine, or Thymine Adenine, Guanine, Cytosine, or Thymine O O O O CH2 O P O P O P O– •• •• OH O O CH2OPO O– O– Polynucleotide chain O– O– Fig. 26.9: Chain Elongation OH Adenine, Guanine, Cytosine, or Thymine O CH2 Adenine, Guanine, Cytosine, or Thymine O O O O –O P O P O– O– O– P O– •• O •• O O CH2OPO O– Polynucleotide chain 26.11 Ribonucleic Acids DNA and Protein Biosynthesis According to Crick, the "central dogma" of molecular biology is: "DNA makes RNA makes protein." Three kinds of RNA are involved. Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) There are two main stages. Transcription Translation Transcription In transcription, a strand of DNA acts as a template upon which a complementary RNA is biosynthesized. This complementary RNA is messenger RNA (mRNA). Mechanism of transcription resembles mechanism of DNA replication. Transcription begins at the 5' end of DNA and is catalyzed by the enzyme RNA polymerase. Fig. 26.10: Transcription Only a section of about 10 base pairs in the DNA is unwound at a time. Nucleotides complementary to the DNA are added to form mRNA. The Genetic Code The nucleotide sequence of mRNA codes for the different amino acids found in proteins. There are three nucleotides per codon. There are 64 possible combinations of A, U, G, and C. The genetic code is redundant. Some proteins are coded for by more than one codon. U U UUU UUC UUA UUG Phe Phe Leu Leu C UCU UCC UCA UCG Ser Ser Ser Ser A UAU UAC UAA UAG C First letter A Second letter Third letter G Table 26.4 Tyr Tyr Stop Stop G UGU UGC UGA UCG Cys Cys Stop Trp U C A G U C A G U C A G U C A G U UUU UUC U UUA UUG CUU CUC C CUA CUG AUU AUC A AUA AUG GUU GUC G GUA GUG Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Ile Met Val Val Val Val C UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala A UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG Tyr Tyr Stop Stop His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu G UGU UGC UGA UCG CGU CGC CGA CCG AGU AGC AGA ACG GGU GGC GGA GCG Cys Cys Stop Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly U C A G U C A G U C A G U C A G U C A G U UAA, UGA, and UAG C U are "stop" codons that UAA Stop UGA Stop A signal the end of the UAG Stop G polypeptide chain. U C C A G AUU Ile ACU Thr AAU Asn AGU Ser U AUC Ile ACC Thr AAC Asn AGC Ser C A AUA Ile ACA Thr AAA Lys AGA Arg A AUG Met ACG Thr AAG Lys ACG Arg G U AUG is the "start" codon. Biosynthesis of all proteins begins with methionine as the first amino C G acid. This methionine is eventually removed after A G protein synthesis is complete. Transfer tRNA There are 20 different tRNAs, one for each amino acid. Each tRNA is single stranded with a CCA triplet at its 3' end. A particular amino acid is attached to the tRNA by an ester linkage involving the carboxyl group of the amino acid and the 3' oxygen of the tRNA. Transfer RNA Example—Phenylalanine transfer RNA One of the mRNA codons for phenylalanine is: 5' UUC 3' The complementary sequence in tRNA is called the anticodon. 3' AAG 5' Fig. 26.11: Phenylalanine tRNA Ribosomal RNA Most of the RNA in a cell is ribosomal RNA. Ribosomes are the site of protein synthesis. They are where translation of the mRNA sequence to an amino acid sequence occurs. Ribosomes are about two-thirds RNA and onethird protein. It is believed that the ribosomal RNA acts as a catalyst—a ribozyme. 26.12 Protein Biosynthesis Protein Biosynthesis During translation the protein is synthesized beginning at its N-terminus. mRNA is read in its 5'-3' direction. Begins at the start codon AUG Ends at stop codon (UAA, UAG, or UGA) Fig. 26.12: Translation Methionine at N-terminus is present as its N-formyl derivative. Reaction that occurs is nucleophilic acyl substitution. Ester is converted to amide. Fig. 26.12: Translation Fig. 26.12: Translation Ester at 3' end of alanine tRNA is Met-Ala. Process continues along mRNA until stop codon is reached. 26.13 AIDS AIDS Acquired Immune Deficiency Syndrome More than 22 million people have died from AIDS since disease discovered in 1980s. Now fourth leading cause of death worldwide and leading cause of death in Africa (World Health Organization). HIV Virus responsible for AIDS in people is Human Immunodeficiency Virus (HIV). Several strains of HIV designated HIV-1, HIV-2, etc. HIV is a retrovirus. Genetic material is RNA, not DNA. HIV HIV inserts its own RNA and an enzyme (reverse transcriptase) in T4 lymphocyte cell of host. Reverse transcriptase catalyzes the formation of DNA complementary to the HIV RNA. HIV reproduces and eventually infects other T4 lympocytes. Ability of T4 cells to reproduce decreases, interfering with bodies ability to fight infection. AIDS Drugs AZT and ddI are two drugs used against AIDS that delay onset of symptoms. O O H3 C N NH HOCH2 O N3 AZT N O HOCH2 N N O H H H H ddI NH AIDS Drugs Protease inhibitors are used in conjunction with other AIDS drugs. Several HIV proteins are present in the same polypeptide chain and must be separated from each other in order to act. Protease inhibitors prevent formation of HIV proteins by preventing hydrolysis of polypeptide that incorporates them. 26.14 DNA Sequencing DNA Sequencing Restriction enzymes cleave the polynucleotide to smaller fragments. These smaller fragments (100-200 base pairs) are sequenced. The two strands are separated. DNA Sequencing Single stranded DNA divided in four portions. Each tube contains adenosine, thymidine, guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs. OH OH OH HO P O O P O O base POCH2 O O HO H DNA Sequencing The first tube also contains the 2,'3'-dideoxy analog of adenosine triphosphate (ddATP); the second tube the 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), the third contains ddGTP, and the fourth ddCTP. OH OH HO P O O P O OH O base POCH2 O O H H DNA Sequencing Each tube also contains a "primer", a short section of the complementary DNA strand, labeled with radioactive phosphorus (32P). DNA synthesis takes place, producing a complementary strand of the DNA strand used as a template. DNA synthesis stops when a dideoxynucleotide is incorporated into the growing chain. DNA Sequencing The contents of each tube are separated by electrophoresis and analyzed by autoradiography. There are four lanes on the electrophoresis gel. Each DNA fragment will be one nucleotide longer than the previous one. Figure 26.13 Figure 26.13 26.15 The Human Genome Project Human Genome Project In 1988 National Research Council (NRC) recommended that the U.S. undertake the mapping and sequencing of the human genome. International Human Genome Sequencing Consortium (led by U.S. NIH) and Celera Genomics undertook project. Originally competitors, they agreed to coordinate efforts and published draft sequences in 2001. 26.16 DNA Profiling and the Polymerase Chain Reaction DNA Profiling DNA sequencing involves determining the nucleotide sequence in DNA. The nucleotide sequence in regions of DNA that code for proteins varies little from one individual to another, because the proteins are the same. Most of the nucleotides in DNA are in "noncoding" regions and vary significantly among individuals. Enzymatic cleavage of DNA give a mixture of polynucleotides that can be separated by electrophoresis to give a "profile" characteristic of a single individual. PCR When a sample of DNA is too small to be sequenced or profiled, the polymerase chain reaction (PCR) is used to make copies ("amplify") portions of it. PCR amplifies DNA by repetitive cycles of the following steps. 1. Denaturation 2. Annealing ("priming") 3. Synthesis ("extension" or "elongation") Figure 26.14: (PCR) (a) Consider double-stranded DNA containing a polynucleotide sequence (the target region) that you wish to amplify. Target region (b) Heating the DNA to about 95°C causes the strands to separate. This is the denaturation step. Figure 26.14: (PCR) (c) Cooling the sample to ~60°C causes one primer oligonucleotide to bind to one strand and the other primer to the other strand. This is the annealing step. Figure 26.14: (PCR) (c) Cooling the sample to ~60°C causes one primer oligonucleotide to bind to one strand and the other primer to the other strand. This is the annealing step. (d) In the presence of four DNA nucleotides and the enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesis step and is carried out at 72°C. Figure 26.14: (PCR) This completes one cycle of PCR. (d) In the presence of four DNA nucleotides and the enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesis step and is carried out at 72°C. Figure 26.14: (PCR) This completes one cycle of PCR. (e) The next cycle begins with the denaturation of the two DNA molecules shown. Both are then primed as before. Figure 26.14: (PCR) (f) Elongation of the primed fragments completes the second PCR cycle. Figure 26.14: (PCR) (f) Elongation of the primed fragments completes the second PCR cycle. (g) Among the 8 DNAs formed in the second cycle are two having the structure shown. Figure 26.14: (PCR) The two contain only the target region and and are the ones that increase disproportionately in subsequent cycles. (g) Among the 8 DNAs formed in the second cycle are two having the structure shown. Table 26.5 Cycle Total DNAs 0 (start) 1 1 2 2 4 3 8 4 16 5 32 10 1,024 20 1,048,566 30 1,073,741,824 Contain only target 0 0 0 2 8 22 1,004 1,048,526 1,073,741,764 26.17 Recombinant DNA Technology