Chapter 26 Nucleosides, Nucleotides, and Nucleic Acids Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

26.1 Pyrimidines and Purines

Pyrimidines and Purines In order to understand the structure and properties of DNA and RNA, one needs to look at the structural components. One set of important components are 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 nitrogen- containing heterocyclic aromatic compounds. PyrimidinePurine N N NHNH N N N

Pyrimidines and Purines Amino-substituted derivatives of pyrimidine and purine have the structures expected from their names, existing as -NH 2. 4-Aminopyrimidine6-Aminopurine H N N N N H H NH2NH2 N N H H HH2NH2N

Pyrimidines and Purines But hydroxy-substituted pyrimidines exist in the keto, rather than the enol, form. However, the rings are still aromatic. enol N N H H HHOHO N N H H HO H keto

Pyrimidines and Purines The same is true for hydroxy-substituted purines which exist in keto, rather than enol, form. H N N N N H H OHOH enol keto H N N N H H O H N

Important Pyrimidines The pyrimidines that occur in DNA are cytosine and thymine. The pyrimidines that occur in RNA are cytosine and uracil. HNHN NHNH O O Uracil HNHN NHNH O O CH 3 Thymine HNHN NHNH NH 2 O Cytosine

Important Purines Adenine and guanine are the principal purines of both DNA and RNA. Adenine N N NH2NH2 N NHNH Guanine O HNHN NHNH N N H2NH2N

Caffeine and Theobromine Caffeine (coffee) and theobromine (coffee and tea) are other naturally occurring purines. Caffeine N N O N N H3CH3C O CH 3 Theobromine O HNHN N N N CH 3 O

26.2 Nucleosides

Nucleosides The classical structural definition of a nucleoside is a pyrimidine or purine N-glycoside of D -ribofuranose or 2-deoxy- D -ribofuranose. Base + Ribose = nucleoside 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.

NH2NH2 HOOH O HOCH 2 O N N Table 26.2 Pyrimidine nucleosides Cytidine occurs in RNA; its 2'-deoxy analog occurs in DNA. Cytidine Note the  - glycosidic bond.

O N NHNH O HO O H3CH3C HOCH 2 Table 26.2 Pyrimidine nucleosides Thymidine occurs in DNA. Thymidine

Table 26.2 Pyrimidine nucleosides HOCH 2 O N NHNH O OHHO O Uridine occurs in RNA. Uridine

Table 26.2 Purine nucleosides Adenosine Adenosine occurs in RNA; its 2'-deoxy analog occurs in DNA. HOCH 2 O OHHO N N N NH2NH2 N

Table 26.2 Purine nucleosides Guanosine Guanosine occurs in RNA; its 2'-deoxy analog occurs in DNA. HOCH 2 N NHNH O O HO N NH2NH2 N OH

26.3 Nucleotides Nucleotides are phosphoric acid esters of nucleosides.

Adenosine 5'-Monophosphate (AMP) Adenosine 5'-monophosphate (AMP) is also called 5'-adenylic acid. Note that the primed numbers are on the sugar ring. OCH 2 P HO O O OHHO N N N NH 2 N 5' 1' 2'3' 4'

Adenosine Diphosphate (ADP) O P O HO OCH 2 P O HO O OHHO N N N NH 2 N Adenosine 5'-diphosphate (ADP).

Adenosine Triphosphate (ATP) O P O HO O OCH 2 P O HO O OHHO N N N NH 2 N P O HO ATP is an important molecule in several biochemical processes including: energy storage (Sections ) and phosphorylation.

ATP and Phosphorylation ATP + hexokinase This is the first step in the metabolism of glucose. ADP + O OH HO (HO) 2 POCH 2 O HOCH 2 O OH HO

3',5'-cAMP and 3',5'-cGMP Cyclic adenosine monophosphate (cAMP) CH 2 O OH N N N NH 2 N O P HO O O Cyclic AMP and cyclic GMP are "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP.

O P HO O O cAMP and cGMP Cyclic guanosine monophosphate (cGMP) CH 2 N NH O O N NH 2 N OH

26.4 Bioenergetics

Bioenergetics Bioenergetics is the thermodynamics of biological processes. Emphasis is on free energy changes (  G). When  G is negative, reaction is spontaneous in the direction written. When  G is positive, reaction is not spontaneous in direction written. When  G is 0, reaction is at equilibrium.

Standard Free Energy (  G°) Sign and magnitude of  G 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).  G in the standard state is called the standard free-energy change and given the symbol  G°. mA(aq)nB(aq)

Standard Free Energy (  G°) Exergonic: An exergonic reaction is one for which the sign of  G° is negative. Endergonic: An exergonic reaction is one for which the sign of  G° is positive. mA(aq)nB(aq)

Standard Free Energy (  G°) 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  G°'. mA(aq)nB(aq)

26.5 ATP and Bioenergetics

Hydrolysis of ATP  G°' for hydrolysis of ATP to ADP is –31 kJ/mol. Relative to ADP + HPO 4 2–, ATP is a "high- energy" 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. ATP + H 2 O ADP + HPO 4 2–

Glutamic Acid to Glutamine + NH 4 + – OCCH 2 CH 2 CHCO – + NH 3 OO + H 2 O H 2 NCCH 2 CH 2 CHCO – + NH 3 OO  G°' = +14 kJ Reaction is endergonic.

Glutamic Acid to Glutamine + NH 4 + – OCCH 2 CH 2 CHCO – + NH 3 OO Reaction becomes exergonic when coupled to the hydrolysis of ATP. + ATP + HPO 4 2– H 2 NCCH 2 CH 2 CHCO – + NH 3 OO  G°' = –17 kJ + ADP

Glutamic Acid to Glutamine – OCCH 2 CH 2 CHCO – + NH 3 OO Mechanism: step 1 involves phosphorylation of glutamic acid. + ATP OCCH 2 CH 2 CHCO – + NH 3 OO + ADPP O –O–O –O–O A mixed anhydride.

Glutamic Acid to Glutamine Step 2: reaction of the phosphorylated glutamic acid with ammonia. H 2 NCCH 2 CH 2 CHCO – + NH 3 OO + HPO 4 2– OCCH 2 CH 2 CHCO – + NH 3 OO P O –O–O –O–O

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 The trinucleotide ATG phosphodiester linkages between 3' of one nucleotide and 5' of the next. A T G free 5' end free 3' end

26.7 Nucleic Acids Nucleic acids are polynucleotides.

Nucleic Acids Nucleic acids were 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 Adenine (A) 30.3%Thymine (T) 30.3% Guanine (G) 19.5%Cytosine (C) 19.9% Total purines: 49.8%Total pyrimidines: 50.1% For example: PurinePyrimidine

Structure of DNA James D. Watson and Francis H. C. Crick proposed a structure for DNA in 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 AT

Base Pairing Ball and stick models showing the Watson and Crick base pairing in A=T.

Base Pairing Likewise, the amounts of G and C in DNA were equal because of complementary hydrogen bonding. 2-deoxyribose GC

Base Pairing Ball and stick models showing the Watson and Crick base pairing in G≡C.

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.

Fig Two antiparallel strands of DNA are paired by hydrogen bonds between purine and pyrimidine bases.

Fig 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 due to the phosphate linkages. DNA winds around histone proteins to form nucleosomes. Nucleosome=Histone proteins+ Supercoiled DNA

Histones Each nucleosome contains one and three-quarters turns of coil = 146 base pairs. Linker contains about 50 base pairs.

26.10 Replication of DNA: Chain Elongation

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. The chain elongates from 5' to 3'.

Fig. 26.9: Chain Elongation OH CH 2 O OP O O– OP O OP O O–O– Adenine, Guanine, Cytosine, or Thymine OH CH 2 OPO O Adenine, Guanine, Cytosine, or Thymine O O– Poly- nucleotide chain 5' end 3' end

Fig. 26.9: Chain Elongation OH CH 2 O OP O–O– Adenine, Guanine, Cytosine, or Thymine O CH 2 OPO O Adenine, Guanine, Cytosine, or Thymine O O– Poly- nucleotide chain O OP O O– OP O O–O– – 5' end 3' end

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.

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.

UUUPheUCUSer UAUTyrUGUCysU UUCPheUCCSerUACTyrUGCCysC UUALeuUCASerUAAStopUGAStopA UUGLeuUCGSerUAGStopUCGTrpG U C A G U C A G U C A G UCAG U C A G First letter Second letter Third letter Table 26.4

UUUPheUCUSer UAUTyrUGUCysU UUCPheUCCSerUACTyrUGCCysC UUALeuUCASerUAAStopUGAStopA UUGLeuUCGSerUAGStopUCGTrpG CUULeuCCUPro CAUHisCGUArgU CUCLeuCCCProCACHisCGCArgC CUALeuCCAProCAAGlnCGAArg A CUGLeuCCGProCAGGlnCCGArg G AUUIleACUThr AAUAsnAGUSer U AUCIleACCThrAACAsnAGCSer C AUAIleACAThrAAALysAGAArg A AUGMetACGThrAAGLysACGArg G GUUValGCUAla GAUAspGGUGly U GUCValGCCAlaGACAspGGCGly C GUAValGCAAlaGAAGluGGAGly A GUGValGCGAlaGAGGluGCGGly G UCAG U C A G

U C UAAStopUGAStopA UAGStopG U C A G AUUIleACUThr AAUAsnAGUSer U AUCIleACCThrAACAsnAGCSer C AUAIleACAThrAAALysAGAArg A AUGMetACGThrAAGLysACGArg G U C A G UCAG U C A G AUG is the "start" codon. Biosynthesis of all proteins begins with methionine as the first amino acid. This methionine is eventually removed after protein synthesis is complete. UAA, UGA, and UAG are "stop" codons that signal the end of the polypeptide chain.

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: UUC 5'3' AAG 3'5' The complementary sequence in tRNA is called the anticodon.

Fig : 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 one- third 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)

26.14 DNA Sequencing The Sanger Method

DNA Sequencing Restriction enzymes cleave the polynucleotide to smaller fragments. These smaller fragments ( base pairs) are sequenced. First the two strands are separated.

DNA Sequencing Single stranded DNA divided in four portions. Each protion contains adenosine, thymidine, guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs. POCH 2 OH O O O P O P O HO base HHOHO O

DNA Sequencing To the first portion, 2,'3'-dideoxyadenosine triphosphate (ddATP) is added; to the second portion, the 2,'3'-dideoxythymidine triphosphate (ddTTP) is added; to the third, ddGTP; and to the fourth, ddCTP. POCH 2 OH O O O P O P O HO base HH O 2,'3'-dideoxynucleoside triphosphate (ddNTP)

DNA Sequencing Each tube also contains a "primer", a short section of the complementary DNA strand, labeled with radioactive phosphorus ( 32 P). 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

End of Chapter 26 Nucleosides, Nucleotides, and Nucleic Acids