28-1 William H. Brown Beloit College William H. Brown Christopher S. Foote Brent L. Iverson Eric Anslyn Chapter 28 Nucleic Acids

28-2  Nucleic acid:  Nucleic acid: A biopolymer containing three types of monomer units. Heterocyclic aromatic amine bases derived from purine and pyrimidine. The monosaccharides D-ribose or 2-deoxy-D-ribose Phosphoric acid.  Following are names and one-letter abbreviations for the five heterocyclic aromatic amine bases most common to nucleic acids

28-3 Purine/Pyrimidine Bases

28-4 Nucleosides  Nucleoside:  Nucleoside: A building block of nucleic acids, consisting of D-ribose or 2-deoxy-D-ribose bonded to a heterocyclic aromatic amine base by a  -glycosidic bond.

28-5 Nucleotides  Nucleotide:  Nucleotide: A nucleoside in which a molecule of phosphoric acid is esterified with an -OH of the monosaccharide, most commonly either the 3’ or the 5’ OH.

28-6 Nucleotides Example 28.1 Example 28.1 Identify these nucleotides.

28-7 Acyclovir & AZT

28-8 DNA - 1° Structure  Deoxyribonucleic acids (DNA) A backbone of alternating units of 2-deoxy-D-ribose and phosphate in which the 3’-OH of one 2-deoxy-D- ribose is joined by a phosphodiester bond to the 5’-OH of another 2-deoxy-D-ribose.  Primary Structure:  Primary Structure: The sequence of bases along the pentose-phosphodiester backbone of a DNA molecule (or an RNA molecule). Read from the 5’ end to the 3’ end.

28-9 DNA - 1° Structure A structural formula for TG phosphorylated at the 5’ end.

28-10 DNA - 2° Structure Base composition in mole-percent of DNA for several organisms.

28-11 DNA - 2° Structure  Secondary structure:  Secondary structure: The ordered arrangement of nucleic acid strands.  The double helix model of DNA 2° structure was proposed by James Watson and Francis Crick in  Double helix:  Double helix: A type of 2° structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right- handed manner about the same axis.

28-12 Base Pairing Base-pairing between adenine and thymine (A-T) and guanine and cytosine (G-C).

28-13 Double Helix Ribbon model of double-stranded B-DNA.

28-14 Double Helix An idealized model of B-DNA.

28-15 Forms of DNA  B-DNA the predominant form in dilute aqueous solution. a right-handed helix pm thick with 3400 pm per ten base pairs. minor groove of 1200pm and major groove of 2200 pm.  A-DNA a right-handed helix, but thicker than B-DNA pm per 10 base pairs.  Z-DNA a left-handed double helix.

28-16 DNA - 3° Structure  Tertiary structure:  Tertiary structure: The three-dimensional arrangement of all atoms of a double-stranded DNA, commonly referred as supercoiling.  Circular DNA:  Circular DNA: A type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by phosphodiester bonds.  Histone:  Histone: A protein, particularly rich in the basic amino acids lysine and arginine, that is found associated with DNA molecules.

28-17 DNA - 3° Structure  Figure Relaxed and supercoiled DNA.

28-18 DNA - 3° Structure  Chromatin:  Chromatin: Consists of DNA molecules wound around particles of histones in a beadlike structure. Further coiling produces the dense chromatin found in nuclei of plant and animal cells.

28-19 Ribonucleic Acids (RNA)  RNA are similar to DNA in that they, too, consist of long, unbranched chains of nucleotides joined by phosphodiester groups between the 3’-OH of one pentose and the 5’-OH of the next; However, the pentose unit in RNA is  -D-ribose rather than  -2- deoxy-D-ribose. the pyrimidine bases in RNA are uracil and cytosine rather than thymine and cytosine. RNA is single stranded rather than double stranded.

28-20 rRNA  RNA molecules are classified according to their structure and function:  Ribosomal RNA (rRNA):  Ribosomal RNA (rRNA): A ribonucleic acid found in ribosomes, the site of protein synthesis.

28-21 tRNA  Transfer RNA (tRNA):  Transfer RNA (tRNA): A ribonucleic acid that carries a specific amino acid to the site of protein synthesis on ribosomes.

28-22 mRNA  Messenger RNA (mRNA):  Messenger RNA (mRNA): A ribonucleic acid that carries coded genetic information from DNA to the ribosomes for the synthesis of proteins. Present in cells in relatively small amounts and very short-lived. Single stranded. Their synthesis is directed by information encoded on DNA. A complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3’ end.

28-23 mRNA The synthesis of mRNA from DNA is called transcription.

28-24 The Genetic Code

28-25  Properties of the Code Only 61 triplets code for amino acids; the remaining 3 (UAA, UAG, and UGA) signal chain termination. The code is degenerate, which means that several amino acids are coded for by more than one triplet. Leu, Ser, and Arg, for example, are each coded for by six triplets. For the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU. There is no ambiguity in the code; each triplet codes for one and only one amino acid.

28-26 Sequencing DNA  Restriction endonuclease:  Restriction endonuclease: An enzyme that catalyzes hydrolysis of a particular phosphodiester bond within a DNA strand. Over 1000 endonucleases have been isolated and their specificities determined. Typically they recognize a set sequence of nucleotides and cleave the DNA at or near that particular sequence. EcoRI from E. coli, for example, cleaves as shown.

28-27 Sequencing DNA Following are several more examples of endonucleases and their specificities.

28-28 Sequencing DNA  Polyacrylamide gel electrophoresis:  Polyacrylamide gel electrophoresis: A technique so sensitive that it is possible to separate nucleic acid fragments differing from one another in only a single nucleotide. Maxam-Gilbert method:Maxam-Gilbert method: A method developed by Allan Maxam and Walter Gilbert; depends on base-specific chemical cleavage. Dideoxy chain termination method:Dideoxy chain termination method: Developed by Frederick Sanger. Gilbert and Sanger shared the 1980 Nobel Prize for biochemistry for their “development of chemical and biochemical analysis of DNA structure.”

28-29 Replication in Vitro During replication, the sequence of nucleotides in one strand is copied as a complementary strand to form the second strand of a double-stranded DNA. Synthesis is catalyzed by DNA polymerase. DNA polymerase will catalyze synthesis in vitro using single-stranded DNA as a template, provided that (1) the four deoxynucleotide triphosphate (dNTP) monomers and (2) a primer are present. Primer:Primer: an oligonucleotide capable of forming a short section of double-stranded DNA (dsDNA) by base- pairing with its complement on a single-stranded DNA (ssDNA).

28-30 Dideoxy Chain Termination The key to the chain termination method is addition to the synthesizing medium of a 2’,3’-dideoxynucleotide triphosphate (ddNTP). Because a ddNTP has no 3’-OH, chain synthesis is terminated where a ddNTP becomes incorporated.

28-31 Dideoxy Chain Termination In this method, the following are mixed: Single-stranded DNA of unknown sequence and primer; then divided into four reaction mixtures.  To each reaction mixture is then added: The four dNTP, one of which is labeled in the 5’ end with phosphorus-32. DNA polymerase. one of the four ddNTPs.

28-32 Dideoxy Chain Termination After gel electrophoresis of each reaction mixture a piece of film is placed over the gel. Gamma rays released by P-32 darken the film and create a pattern of the resolved oligonucleotide. The base sequence of the complement to the original strand is read directly from bottom to top of the developed film.

28-33 Dideoxy Chain Termination The primer-DNA template is divided into four separate reaction mixtures. To each is added the four dNTPs, DNA polymerase, and the four ddNTPs.

28-34 Dideoxy Chain Termination The mixture is separated by polyacrylamide gel electrophoresis.

28-35 Nucleic Acids End Chapter 28