Nucleic Acid Biochemistry 104 PDF

# Chapter 4 Nucleic Acids ## Recall from Chapter 1 - The names of the principal organic constituents of cells and organisms: proteins, nucleic acids, carbohydrates, and lipids - They make up a large portion of all living matter - Proteins, nucleic acids, and polysaccharides are all polymers of mo...

# Chapter 4 Nucleic Acids ## Recall from Chapter 1 - The names of the principal organic constituents of cells and organisms: proteins, nucleic acids, carbohydrates, and lipids - They make up a large portion of all living matter - Proteins, nucleic acids, and polysaccharides are all polymers of monomeric constituents, held in linear order by hydrolyzable chemical bonds - Lipid molecules differ in not being polymeric, lipids usually exist as components of giant complexes - membranes and lipoproteins ## The Nature of Nucleic Acids - Nucleic acids play roles in the **storage and transmission of biological information** - They are considered the **most fundamental of biological molecules** - Life began with nucleic acids because only they, of all biological substances, have the potential for **self-duplication** - The blueprint for an organism is encoded in its nucleic acid - Proteins that cells will make and their functions are recorded in these molecules - Nucleic acids preserve and transmit genetic information - They are involved in: - DNA replication - Transcription - Translation ## The Two Types of Nucleic Acid: DNA and RNA - **DNA** was discovered in 1869 by Friedrich Miescher, a military surgeon during the Franco-Prussian War - Miescher discovered an acidic substance in the pus from discarded surgical dressings - the material was predominantly in the nuclei of the white blood cells - DNA was named *nukleinsäure* or nucleic acid - Nucleic acids are composed of: - **Organic nitrogenous bases** - **A pentose sugar** - **Phosphate** - **DNA** is **deoxyribonucleic acid** - The other major form is **RNA**, **ribonucleic acid** - Each is a polymeric chain in which the **monomer units are connected by covalent bonds** ## The Structure of Monomer Units of RNA and DNA - The monomer units of RNA and DNA are shown in the following table: | Molecule | Phosphate | 2'-Deoxyribose | Base | |---|---|---|---| | Repeating unit of ribonucleic acid (RNA) | O-P=O <br> Ο <br> CH₂ <br> 5 <br> C <br> 2 <br> Base <br> Ribose <br> C <br> 4°C <br> H <br> H <br> C <br> 2' <br> H <br> C <br> 3 <br> H <br> C <br> 2 <br> O <br> H <br> <br> HICIO <br> H <br> C <br> 1 <br> H <br> C <br> 2 <br> H <br> <br> <br> OH | | <br> 5CH2 <br> Base <br> 2'-Deoxyribose <br> C <br> H <br> C <br> 2 <br> H <br> C <br> 3 <br> H <br> C <br> 2 <br> HC <br> H <br> C <br> 1 <br> H <br> C <br> 2 <br> H <br> <br> <br> <br> H | | | Repeating unit of deoxyribonucleic acid (DNA) | <br> O-P=O <br> Ο <br> CH₂ <br> 5 <br> C <br> 2 <br> Base <br> Ribose <br> C <br> 4°C <br> H <br> H <br> C <br> 2' <br> H <br> C <br> 3 <br> H <br> C <br> 2 <br> O <br> H <br> <br> HICIO <br> H <br> C <br> 1 <br> H <br> C <br> 2 <br> H <br> <br> <br> <br> OH | | <br> 5CH2 <br> Base <br> 2'-Deoxyribose <br> C <br> H <br> C <br> 2 <br> H <br> C <br> 3 <br> H <br> C <br> 2 <br> HC <br> H <br> C <br> 1 <br> H <br> C <br> 2 <br> H <br> <br> <br> <br> H | | - The difference between ribose and deoxyribose lies solely in the **2' hydroxyl group on ribose in RNA, which is replaced by hydrogen in DNA** - **Each successive monomer unit in the nucleic acids is connected by a phosphate group attached to carbon 5' of one unit and carbon 3' of the next one** - This forms a **phosphodiester link between two sugar residues** - The link is named this way because hydrolysis of this phosphate diester link yields one acid (phosphoric acid) and two alcoholic sugar hydroxyls. - Long nucleic acid chains are built up this way, containing up to hundreds of millions of units. - Each nucleic acid contains a **heterocyclic base** linked to the 1' carbon of the sugar ## The Composition and Structure of Nucleic Acids - Both DNA and RNA are **polynucleotides** - **RNA** contains the **sugar ribose** - **DNA** contains **deoxyribose** - The **nucleic acid bases** come in two kinds: - **Purines**: adenine and guanine - **Pyrimidines**: cytosine, thymine, and uracil - **RNA and DNA use three of the same bases**, with uracil being used by RNA, where DNA uses thymine. - See Figure 4.2 for structures of the purines and pyrimidines. - The **phosphate group is a strong acid** with a pK of about 1. - The **phosphodiester-linked sugar residues form the backbone** of the nucleic acid molecule. - The backbone is a repetitive structure and **incapable of encoding information** - The **importance of the nucleic acids in information storage and transmission derives from their being heteropolymers** - Each monomer in the chain carries a heterocyclic base - The following bases are found in DNA: - **Adenine (A)** - **Guanine (G)** - **Cytosine (C)** - **Thymine (T)** - RNA contains the same bases as DNA, except **thymine is replaced by uracil (U)** - **Each nucleic acid chain can be regarded as a polymer made from four kinds of monomers**. - These monomers (**nucleotides**) are phosphorylated ribose or deoxyribose molecules with purine or pyrimidine bases attached to their 1' carbons. - In purines the attachment is at nitrogen 9, in pyrimidines at nitrogen 1. - This bond is known as a **glycosidic bond** - The **nucleosides** are **monomers**, each nucleotide can be considered the 5'-monophosphorylated derivative of a sugar-base adduct. - These nucleotides are also called **nucleoside 5'-monophosphates**. - See Figure 4.3 for structures of nucleosides and nucleotides. ## Properties of the Nucleotides - Nucleotides are **strong acids**, with a primary ionization pKa of approximately 1.0 - The **bases are also capable of conversion between tautomeric forms** - **Tautomeric forms are structural isomers differing only in the location of their hydrogen atoms and double bonds** - The major forms are those shown in Figure 4.2 - The **bases and all of their derivatives absorb light strongly in the ultraviolet region of the spectrum** - The **UV absorbance depends on the pH** because of the ionization reactions in the bases - **Ultraviolet light has chemically damaging effects on DNA** ## Stability and Formation of the Phosphodiester Linkage - A **polynucleotide could be generated from its nucleotide monomers by elimination of a water molecule** between each pair of monomers. - This **hypothetical reaction has a positive free energy change**, which is **about +25 kJ/mol**, so **equilibrium lies far to the side of hydrolysis** of the phosphodiester bond - This illustrates **the metastability of biologically important polymers** - Polynucleotides are **thermodynamically favored to break down**, but they do so only very slowly unless the reaction is catalyzed. - **Polynucleotides should hydrolyze under conditions existing in living cells, but their hydrolysis is exceedingly slow** unless catalyzed - **Hydrolysis of polynucleotides to nucleotides is the thermodynamically favored process** ## Factors Involved in Polynucleotide Formation - The **unfavorable thermodynamics** of the hypothetical dehydration reaction leads to the question of how polynucleotides actually are synthesized - The **synthesis** involves **the energy-rich nucleoside or deoxynucleoside triphosphates** - The basic reaction is simple, instead of the dehydration reaction, the **nucleoside monophosphate being added to the growing chain is presented as a nucleoside triphosphate** (like ATP) ## The Energetics of Polynucleotide Formation - This reaction can be considered the **sum of two reactions** - Hydrolysis of a nucleoside triphosphate - Formation of a phosphodiester link by elimination of water - The **coupled reaction is favorable because the net AG°' is negative** - The reaction **is further favored because the hydrolysis of the pyrophosphate product (PP₁) to orthophosphate, or inorganic phosphate (P₁), has a AG of about -19kJ/mol.** - **Polynucleotide synthesis is an example of a principle we emphasized in Chapter 3 - the use of favorable reactions to drive thermodynamically unfavorable ones.** ## The Primary Structure of Nucleic Acids - Polynucleotides always have a **sense or directionality** - **The phosphodiester linkage between monomer units is between the 3' carbon of one monomer and the 5' carbon of the next** - **One end carries an unreacted phosphate, the other end carries an unreacted 3' hydroxyl group** - Each polynucleotide also has **individuality** - The **nucleotide sequence is called the primary structure of the particular nucleic acid** - The primary nucleotide sequence dictates the **functions of the organism** ## DNA as the Genetic Substance: Early Evidence - Early scientists were split between whether DNA or protein was responsible for genetic information - Oswald Avery, Colin MacLeod, and Maclyn McCarty found that the DNA from pathogenic strains of the bacterium *Pneumococcus* could be transferred into nonpathogenic strains, making them pathogenic - Alfred Hershey and Martha Chase studied infection of the bacterium *Escherichia coli* by a bacterial virus, the bacteriophage, or phage, T2 - They labeled T2 bacteriophage with the radioisotopes 35S and 32P and concluded that when the phage attached to *E. coli*, it was mainly the 32P (and hence the phage DNA) that was transferred into the Bacteria - This resulted in the **general recognition by 1952 that DNA must be the genetic substance** - The question remained of how DNA could carry the enormous amount of information that a cell needed and how it could be accurately replicated ## Secondary and Tertiary Structure of Nucleic Acids - **X-ray diffraction studies** of concentrated DNA solutions showed that DNA in the fibers must have some kind of regular, repetitive three-dimensional structure - Rosalind Franklin photographed the DNA diffraction patterns showing that DNA had a **regular, repetitive three-dimensional structure**. - This structure is called the **secondary structure**. - It is distinguished from the **primary structure**, which is simply the sequence of individual nucleotide residues. - **Watson and Crick** recognized that the DNA fiber diffraction exhibited a cross pattern typical of a helical secondary structure. - Their model consisted of **two antiparallel DNA strands** that were **complementary**. - This meant that **A must always pair with T, and G must always pair with C**. - This also explained **Chargaff's rule**, which stated that A and T were almost always present in nearly equal quantities, as were G and C. ## The Watson-Crick Model for DNA - The key aspect of this model was that **the two strands could be stabilized by hydrogen bonding** between base pairs on opposite strands. - The **distances between the 1' carbons** of the deoxyribose moieties of A-T and of G-C are the same, about 1.1 nm in each case. - **This pairing arrangement meant that the double helix could be regular in diameter** - **The bases are closely packed within the helix** - There are two deep spiral grooves called the **major groove** and the **minor groove** - The **bases can be approached through the major groove**, which gives more direct access to the bases - The **minor groove faces the sugar backbone** ## The Semiconservative Nature of DNA Replication - If the strands of a DNA molecule could be separated, **new DNA could be synthesized along each**, following the same base-pairing rule. - **Two double-stranded DNA molecules would be obtained, each an exact copy of the original** - This process of **self-replication is precisely the property that the genetic material must have**. - This is how **two complete copies of the genetic information carried in the original cell must be produced** when a cell divides. - **The replicative hybrid contains one complete strand of parental DNA and one complete strand of newly synthesized DNA.** ## Alternative Nucleic Acid Structures: B and A Helices - X-ray diffraction studies had already been obtained for DNA, indicating that the molecule can exist in more than one form. - The **B form**, which is seen in DNA fibers prepared under conditions of high humidity, is the most common form in cells. - The **A form** is seen in DNA fibers prepared under conditions of low humidity - RNA molecules always form the A structure, as do DNA-RNA hybrid molecules. ## Properties of A- and B-Form DNA - Both are right-handed helices, but the A form lies further out from the helix axis - The bases are strongly tilted with respect to the helix axis. - There is a difference in the surfaces of the A and B Forms, with the major and minor grooves being more distinct in B-Form DNA. - Scientists have succeeded in crystallizing a small double-stranded DNA fragment, which allowed for a more accurate determination of DNA structures. ## Stability and Formation of the Phosphodiester Linkage - The major polynucleotide secondary structures (the A and B forms) are relatively stable under physiological conditions. Yet they must not be too stable because important biochemical processes-DNA replication and transcription-require that the double-helix structure be opened up. - **When it extends over large regions this loss of secondary structure is called denaturation** ## The Factors Favoring Denaturation - Two major factors favor dissociation of double helices into randomly coiled single chains: - **Electrostatic repulsion between the chains**: every nucleotide carries a negative charge - **The higher entropy of the random-coil structure**, which has many possible configurations - The free energy change in going from a regular two-stranded polynucleotide secondary structure (such as B-form DNA) to individual random-coil strands is given by the usual formula: AG = ΔΗ - ΤTAS (helix random coil) - **In order for the double helix to be stable under any conditions, AG for the unfolding reaction must be positive** - **Therefore, we must look for a large contribution from AH to compensate for the factors just mentioned** - The **sources of such a positive AH are the hydrogen bonds between the base pairs and van der Waals interactions between stacked bases.** ## Superhelical Energy and Changes of DNA Conformation - The amount of free energy stored in supercoiling (AGsc) is proportional to the square of the superhelical density σ: AGS = Ko^2 - **Highly supercoiled DNA has a lot of stored energy, which can be reduced by any process that decreases the superhelix density** ## The Role of Superhelical stress in DNA Structure - The **superhelical stress can promote any one of the following changes** in DNA: - Local melting - Z-DNA formation - Cruciform extension - Formation of H-DNA regions ## SUMMARY - **DNA** is a **deoxyribonucleic acid** (DNA) Each is a polynucleotide, a polymer of **four kinds of nucleoside 5'-phosphates**, connected by links between 3' hydroxyls and 5' phosphates - **RNA** is a **ribonucleic acid** (RNA) with the sugar ribose - **DNA** has deoxyribose - The **phosphodiester linkage** connecting these monomers is inherently unstable, but only hydrolyzes slowly in the absence of catalysts - **Early evidence suggested that DNA might be the genetic material**, but it was not until Watson and Crick elucidated its two-stranded secondary structure in 1953 that it became obvious how DNA might direct its own replication - The structure they proposed involved **specific pairing between A and T and between G and C.** - The **helix is right-handed**, with about 10.5 base pairs (bp) per turn in the B form. Such a structure can replicate in a **semiconservative manner**, as demonstrated by Meselson and Stahl in 1958. - **Other forms of polynucleotide structures exist, of which the most important is the A form, found in RNA-RNA and DNA-RNA double helices** - **Most DNA is double-stranded in vivo** - **Circular DNA can exist in a variety of topologies** - **Most circular DNA molecules are supercoiled** - **Most RNA is single-stranded**, but it may fold back to form hairpins and other well-defined tertiary structures - **DNA contains stored genetic information, which is transcribed into RNA**. - Some of these RNA molecules act as messengers to direct protein synthesis. - The **messenger RNA** is **translated on a ribosome, using the genetic code, to produce proteins** - **Supercoiling of DNA can be expressed in terms of twist (T) and writhe (W)**. - These terms are related to the **linking number** (L) by **L = T + W.** - **To form superhelical coiling requires the expenditure of ATP energy, using an enzyme called DNA gyrase** - Gyrase belongs to a class of **topoisomerases**; others relax supercoiled DNA. - **Polynucleotides can form a number of unconventional structures**, including: - Left-handed DNA (Z-DNA) - Cruciforms - Triple helices - G-quadruplexes. - **The secondary structures of polynucleotides can be changed in various ways** - The helix can “melt,” which involves strand separation. This change is easiest for regions rich in A-T pairs. - **Energy stored in superhelical DNA may promote local DNA melting or changes to a variety of alternative structures**, such as Z-DNA, cruciforms, or a particular triple-helical structure called H-DNA.