Biochem Chapter 10

Biochem Chapter 10 - Concepts in Biochemistry 3rd Edition...

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Unformatted text preview: Concepts in Biochemistry 3rd Edition Chapter Ten DNA and RNA: Structure and Function Dr J. Davis Outline 10.1 RNA and DNA Chemical Structures 10.2 DNA Structural Elements 10.3 RNA Structural Elements 10.4 Cleavages of DNA and RNA by Nucleases 10.5 Nucleic Acid-Protein Complexes 2 Chapter 10 Homework #1 (as needed), 2, 6, 9, 12, 16, 19 21, 29, 31 and 32. Chapter Seven Introduction Two (2) types of nucleic acid biomolecules are responsible for storage, transfer, and expression of genetic information in living systems: 1) DNA [Deoxyribonucleic acid] and 2) RNA [Ribonucleic acid]. DNA is found in chromosomes in the cell nucleus, serving as the repository of genetic information. DNA is also found in Mitochondria and in Chloroplasts [plants]. RNA is present is 3 types: ribosomal, messenger and transfer RNA. Recall: Flow of biological Info: DNA RNA Proteins involved in cell structure/function (Chap. 1).. 4 Introduction Nucleic acids are linear polymers of 4 different monomers called Nucleotides. Like Proteins, DNA has specific sequences and also a specific 3-D structure s well as a specific cell function. These Nucleotides lead to the 20 different amino acids essential for life. This Chapter will explore the chemical and biological properties of DNA/RNA. We will also introduce information on combinations of proteins and nucleic acids (nucleic acid- protein complexes). 5 10.1 RNA and DNA Chemical Structures Nucleotides Nucleic acids are linear polymers of nucleotides. Nucleotides have 3 chemical parts: (1) An aromatic, heterocyclic ring compound called a Nitrogenous base; (2) A five-carbon ribose ring; and (3) one, 2 or 3 phosphate groups. Figure 10.1 shows the general structure of a nucleotide. The Nitrogenous Bases in DNA are derivatives of heterocyclic [C, N, H, O] organic compounds called Purines: Adenine [A] and Guanine [G] and derivatives of organic compounds called Pyrimidines: Thymine [T] and Cytosine [C]. 6 Fig 10.1 General Structure for a Nucleotide 7 10.1 RNA and DNA Chemical Structures Nucleotides (cont.) RNA contains the purines, Adenine & Guanine plus the pyrimidines Cytosine and the base Uracil (instead of Thymine). RNA also contains methylated bases in small quantities [5-methylcytosine, 5-hydroxymethylcytosine, 2methyladenine and 1-methylquanine, etc.] Structures of the major bases in RNA and DNA are shown in Fig 10.2. 8 Figure 10.2 Heterocyclic bases in DNA/RNA 9 10.1 RNA and DNA Chemical Structures Nucleosides: ribose sugar + a nitrogenous base Nucleic acids contain an aldopentose plus a nitrogenous base Two type of sugars exist in nucleic acids: 1) Ribose occurs in RNA; 2) 2-Deoxyribose [lacking an oxygen group on C2 this denotes the only difference between the two forms aldopentose takes], occurs in DNA. The ribose (the sugar) and the nitrogenous base are connected thru an N-glycosidic linkage to form a Nucleoside. The covalent linkage formed by elimination of water, connects thru the anomeric C1 on ribose, and the N9 of purines and N1 of pyrimidines [a -linkage exist]. 10 10.1 RNA and DNA Chemical Structures Nucleosides: ribose sugar + a nitrogenous base A dual numbering system is used to identify C/N atoms in the ring systems of the nucleoside. Structures of the aldopentose sugars and of Nucleosides are shown in Figure 10.3 / 10.4. Note: Many synthetic nucleosides have potent biological physiological activity: Some synthetic nucleoside are used as drugs for treatment of cancers and other diseases (i.e., AZT or DDI for HIV-AIDS; etc). 11 Fig 10.3 Aldopentose Sugars in DNA/RNA Fig. 10.4 Purine and Pyrimidine Nucleoside Structure 12 Fig 10.5 Structures of AZT and DDI 13 10.1 RNA and DNA Chemical Structures Nucleotides : a nucleoside + phosphate A Nucleotide is formed when a phosphate group is added to the ribose sugar hydroxyl group (nucleoside), via ester linkage to C-5' (usually) on the pentose sugars. Addition of 1, 2 or 3 phosphates, forms nucleoside mono-, di-, or tri-phosphates. Products are also called nucleoside 5'- monophosphate or 5'- mononucleotide (1 phosphate). AMP is the most important and most abundant nucleotide in the cell. Figure 10.5 shows structures for three Adenine nucleotides: AMP; ADP; and ATP Nomenclature [LEARN THESE] for the major 14 nucleotides/nucleosides in DNA/RNA are in Table 10.1. 15 Fig 10.6 Three Types of Nucleotides 16 10.1 RNA and DNA Chemical Structures Nucleotide Formation The 1st Phosphate [-] is added via a phosphate ester linkage; subsequent phosphates (- and -) are add via phosphoanhydride linkage (w/o H2O) (Chap. #14). ATP is the main carrier of chemical energy in the cell. ATP + H2O ADP + Pi and Energy 17 10.1 RNA and DNA Chemical Structures Nucleotides Other nucleoside tri-phosphates [GTP, UTP, dGTP; dUTP] are used in many biosynthetic processes including the synthesis of nucleic acids. Nucleoside phosphates also function as important signaling molecules involved in cell communication. : GTP; c-AMP; c-GMP are intermediates involved in the transmission of messages via signal transduction thru cell membranes. They are also constituents of important cofactors, like Coenzyme A, Flavin adenine dinucleotide [FAD] and NAD+. 18 10.1 RNA and DNA Chemical Structures Nucleotides Protons on the nucleotide phosphate groups are relatively acidic, pKa values [~ 2 6]. At physiological pH, the predominant nucleotide structure has all protons dissociated from the phosphates; the compounds all have multiple negative charges. ATP has a net charge of - 4. Cellular nucleotides are usually associated with metal ions such as Mg2+ or Mn2+ to neutralize these charges. Check out the Windows on Biochemistry (10-1) for examples of Purines/pyrimidines with biological activity. 19 10.1 RNA and DNA Chemical Structures Nucleic Acids Nucleic acids are formed from nucleotides linked together thru the 5'- phosphate group of one nucleotide and the 3'hydroxyl group of the next. The linkage is aka 3', 5'-Phosphodiester bond . Some Characteristic properties of Nucleic Acids The covalent backbone of the DNA [and RNA] consists of alternating (deoxy)ribose sugars and phosphate groups. This is a common, invariant part the structure of nucleic acids, which also plays a critical structural role. The covalent backbone has Directionality: One end of chain has a 3'-hydroyxl group; the other has a 5'phosphate [See Figure 10.7]. 20 Fig 10.7 DNA Structure showing the Phosphodiester bond linkages (3'- 5') 5'-end 3'-end 21 10.1 RNA and DNA Chemical Structures Nucleic Acids The nitrogenous bases are linked to the backbone via Nglycosidic bonds and protrude from the structural backbone like side chains [this is the variable region of DNA/RNA]. These non-polar nitrogenous bases are sequestered at right angles, as far as the stereochemistry is concerned, to the ribose backbone. The sequence of the 4 bases carries the specific genetic message. Drawing Nucleic Acid structures Drawing nucleic acids is cumbersome, so one simple way to represent them is shown below: Biochemists use a vertical line to represent the sugars and 22 letters to represent the bases. 10.1 RNA and DNA Chemical Structures The top of the line indicates C1' of the sugar where the base is attached by an N-glycosidic bond; the bottom of the line is labeled C5'. The phosphate is inserted as P and the 3',5'-phosphodiester bonds are shown as connecting lines (See next slide). 23 10.1 RNA and DNA Chemical Structures Structure of an oligonucleotide containing 4 bases. Note: 5'-end (left) and 3'- end (right). An abbreviated nomenclature can be used to represent this sequence in DNA (or RNA): T(U)ACG The oligonucleotide is simply a short sequence of nucleic acids. 24 10.1 RNA and DNA Chemical Structures DNA Structures and Bonding The physical properties of DNA for several different species are compared in Table 10.2 DNA comprises ~1% of total cell weight. DNA from simple prokaryotic cells [E. coli] is a single chromosome = one huge molecule [MW = 2.6 billion Daltons/ 4.7 million base pairs]; ~1.4 mm in length. Eukaryotic DNA Human DNA is large: has ~3 billion base pairs and a total length of 1- 2 meters. Note: DNA is somewhat difficult to isolate from cells. 25 26 10.1 RNA and DNA Chemical Structures RNA (Comprises ~ 5-10% of total weight of a cell). RNA exists in 3 major forms. Each form has a characteristic base composition and molecular size [Table 10.3]. 1. Ribosomal RNA [rRNA] is the most abundant, and is associated with the Ribosomes, protein synthesizing organelles. There are 3 kinds of prokaryotic rRNA: 5S, 16S and 23S. The S stands for the sedimentation coefficient which defines the rate of sedimentation in a centrifugal field; S values depends on the weight, shape and density of the biomolecule. 27 10.1 RNA and DNA Chemical Structures RNA 2. Messenger RNA [mRNA] carries the coding for protein synthesis from nuclear DNA to the ribosomes. Each mRNA molecule carries the coding for at least 1 gene, which in turn codes for at least 1 polypeptide product. 3. Transfer RNA [tRNA], is the smallest nucleic acid [w 70 -90 nucleotides]. tRNA forms esters w. specific amino acids for use in protein synthesis. SEE Table 10.3 RNA molecules in E coli. 28 29 10.1 RNA and DNA Chemical Structures Properties of Nucleic Acids (Covalent Structure) 1. Polarity/Nonpolarity Nucleic acids have acidic and basic regions!! Phosphate groups in the backbone have negative charges (due to proton dissociation at physiological pH). These ionic charges plus the polar sugars form a region of hydrophilic character for nucleic acids which means the backbone interacts favorably with water. The variable region where the nitrogenous bases reside are considered hydrophobic. The nitrogens are weakly basic plus the amino (--NH2) groups are not protonated, so there is little charge in this region. 30 10.1 RNA and DNA Chemical Structures Properties of Nucleic Acids (Covalent Structure) 1. Polarity/Nonpolarity (B) Overall, the polarity differences between the backbone and the bases enhances the folding of the DNA/RNA into 3-D structures. The outer backbone is well suited for the aqueous environment in the cell, while the nonpolar bases move to the inside and, they can interact with each other, w/o any major contact with each other. 2. Hydrogen Bonding Nucleic acid folding is also influenced by Hydrogen bonding between several functional groups on the nonpolar nitrogenous bases. 31 10.1 RNA and DNA Chemical Structures Properties of Nucleic Acids (Covalent Structure) 2. Hydrogen Bonding (Cont.) Ring N--H groups and amino groups [NH2] are H bond donors; Carbonyl groups [>C==O] and ring nitrogen atoms [==N--] are potential H bond acceptors. Hydrogen bonding allows the two strands to associate and fold into the double helix structure as proposed by Watson and Crick. Hydrogen bonding is also is important in the folding of the single stranded RNA molecules. 32 10.2 DNA 3-D Structural Elements Watson & Crick published a paper (1953) which connected all the experimental dots of the elucidation of DNA structure. X-ray Crystallography data for DNA [from Rosalind Franklin] showed regular repeating patterns of the structural units. We now know that DNA consists of polynucleotides arranged in a double helix (in vivo). Many of the same basic noncovalent interactions influence the final structure of nucleic acids as with proteins. The Primary[10] Structure of DNA/RNA consists of an invariant, covalent backbone and sequence variable nitrogen bases. The 20 Structure of nucleic acids (helix) is defined by the repeated [periodic] arrangements of the polynucleotide 33 strands. 34 10.2 DNA 3-D Structural Elements Watson and Crick (1953) used three main sources of information to determine DNA's fine structure: 1) the X-ray crystallography data, 2) chemical analysis data of the ATCG base content [relative concentration ratios of A:T; or G:C], plus, 3) chemical data on base structure (bases exist in ketotautomeric forms) which allowed hydrogen bonding between A:T [= 2 H bonds] and G: C [= 3 H bonds). DNA Double Helix Features DNA consists of two complementary, polynucleotide chains twisted into a helical staircase structure [SEE Figure 10.8]. 1. The two strands are right-handed, helical polynucleotide chains, coiled around a common axis, forming a double helix. The structure includes a major groove and a minor groove. 35 36 10.2 DNA 3-D Structural Elements DNA Double Helix Features 2) The two strands run antiparallel [in opposite directions]. i.e., The 3',5' phosphodiester chains run in opposite directions. 3) In aqueous media, the Polar charged, covalent backbone of alternating deoxyribose sugars and phosphate groups are localized on the outside to interact with water; the hydrophobic purine/pyrimidine bases avoid water by sequestering inside the structure. 4) Double helix is stabilized by: a) Hydrogen Bonds between base pairs, and b) Van der Waals and hydrophobic interactions between stacked bases Note: the bases are close enough do that their pi electrons can overlap (Van der Waals). 37 Fig 10. 9 Hydrogen bonding between base-pairs [A:T (2) and G:C (3)] 38 10.2 DNA 3-D Structural Elements Complementary Base Pairing is the most important feature of the structure This feature is the key to the function of storage and transfer of genetic information in living cells. Complementary base pairing prevents variations in the internal spacing between the polynucleotide chains, allowing only pyrimidine-purine pairing. It cannot accommodate purine-purine or pyrimidine pyrimidine base pairing (too far apart)]. 39 10.2 DNA 3-D Structural Elements Forms of DNA B-DNA is a major observed, DNA conformational or form of DNA isolated from aqueous solutions (retains water in the crystal structure). It is probably the major form in vivo. If DNA is treated with reagents to dehydrate the molecule, the B-DNA structure rearranges to the ADNA form. A-DNA has 11 bases per turn and is shorter and more compact than B-DNA [10.5 bases per turn]. 40 10.2 DNA 3-D Structural Elements Forms of DNA (cont.) Both B- and A-DNA are right-handed helixes, with antiparallel strands. A newer structural form, Z-DNA is observed in short strands of synthetic DNA. It has left handed helix; 12 bases per turn. Z refers to the zigzag structure of backbone. Note: Z-DNA has also been observed in native DNA; it may function in gene regulation(?). 41 Fig 10.10 Comparison of Secondary Structures of A-, B-, and Z-DNA 42 Fig 10.11 A possible scheme for Replication of DNA (See Chap 11). 43 10.2 DNA 3-D Structural Elements Physical and Biological properties of DNA Watson & Crick postulated (10.11) that DNA could separate and unwind to give two complementary strands that act as templates for replication of 2 new strands during the process of replication. Early investigations showed that this was possible Here's the evidence: 1. DNA Unwinding DNA unwinds when exposed to mild conditions, such as: a) Heat, b) acids/bases, or c) organic solvents. The DNA double helix strands separate--a process aka Denaturation [Figure 10.12] When heat is used, we call the process melting (occurs over a small temperature range [85-90 oC]). 44 10.2 DNA 3-D Structural Elements 1) DNA Unwinding (cont.) When melting occurs, there is a significant increase in absorption of UV light by the DNA. This is an experimental technique for quantifying DNA denaturation, and also shows the relative strength of Hbonding and stacking interactions of the nitrogenous base pairs. The increase in absorbance (optical density) of DNA at 260 nM is called a HYPERCHROMIC SHIFT. It is due to changes in the electronic environment around the aromatic bases when an ordered double helix arrangement is disrupted forming a random coil (unpaired DNA strands). 45 Fig 10.12 DNA Denaturation and Renaturation 46 10.2 DNA 3-D Structural Elements 1) DNA Unwinding (cont.) Figure 10.13 shows Melting curves for several DNA molecules from different microorganisms. Plots were obtained by heating DNA and measuring the shift in absorbance at 260 nm (More light is absorbed when DNA is denatured)! The melting temperature, Tm, is measured at the midpoint of the curve. The nature of each curve reflects the content of ACTG in the DNA. Renaturation of free DNA strands is possible (back into the original double helix), if solutions are cooled slowly, a Process is called ANNEALING or Renaturation. 47 Fig 10.13 Melting Curves for 4 different DNA Molecules 48 10.2 DNA 3-D Structural Elements Tertiary Structure of DNA Recall: Secondary structure of DNA is the double helix. There exist two major forms of DNATertiary structure: 1) Circular DNA, and 2) Linear DNA. 1. Circular DNA: Many microorganisms, have a closedcircular DNA [includes some viruses, bacteriophages, and some animals. Closed, circular duplex DNA exists in two forms--1) a relaxed coiled DNA and 2) a supercoiled DNA [10.14]. The formation and interconversion of these two circular DNA forms is done by enzyme proteins called Topoisomerases (b & c.) Topoisomerase enzymes catalyze changes in the topology 49 of DNA. Fig 10.14 Tertiary Forms of DNA: a) Circular, Duplex DNA 50 DNA supercoiling may have biological significance: 1) It makes DNA more compact [for easier storage in the cell], and 2) It may play a regulatory role in DNA replication. 51 10.2 DNA 3-D Structural Elements Tertiary Structure of DNA Quadruplex DNA There is evidence for four-stranded DNA forms aka Quadraplex DNA. Quadraplex DNA [Figure 10.15] has been observed in a protozoan and in a human oncogene. Quadraplex aka G-quadruplexes occurs in Guanine-rich areas of DNA. These structures may play a role in the regulation and stabilizing of telomeres [Chromosome ends], and in regulation of gene expression. Additionally they may limit/ inhibit telomerase action in continuously extending chromosome ends and thus preventing the start of 52 cancer. Figure 10.15 Quadruplex DNA 53 10.3 RNA (3D) Structural Elements Recall: RNA differs from DNA in two major ways: (1) RNA contains Ribose sugar [not deoxyribose], and (2) contains the base Uracil (U) and not Thymine (T). The extra Hydroxyl group on C2 (ribose ring) makes RNA more susceptible hydrolysis than DNA (Why DNA is the more stable nucleic acid for storage of genetic information [in forensic and anthropological samples]). This difference in primary structure also affects RNA's 2 O / 3 O RNA structure. 1. All RNA is single-stranded. RNA exists as a phosphodiester backbone [alterating ribose and phosphate] plus sidearm nitrogenous bases perpendicular to the chain. 54 10.3 RNA (3D) Structural Elements 1. (ss-)RNA structures often loop back onto themselves forming contorted conformations [Figure 10.16]. Structure elements include: 1. HAIRPIN Turns (aka loops) in the single strand. Bring together complementary stretches of base pairing with themselves [pseudo double helix regions similar to A-DNA. 2. Right-Handed Double Helixes result from intra-strand folding. Regions are antiparallel and stabilized by same H bonding interactions as DNA [A::U; G ::: C]. Steric interferences of the C2 hydroxyl group on ssstranded RNA cannot fold into the analogous B-DNA arrangement. 55 10.3 RNA (3D) Structural Elements 1. Internal Loops/Bulges, exist in many RNA molecules and disrupt the formation of continuous double helix regions. 56 Fig. 10.16 ssRNA Secondary and Tertiary Structure. 57 10.3 RNA (3-D) Structural Elements tRNA Structure tRNA is the smallest RNA molecule, and functions as a carrier of specific amino acids used for protein synthesis. Each of the 20 amino acids have at least one assigned tRNA. All tRNAs contain from 74 93 nucleotides in a single chain; including several unusual purine & pyrimidine bases. tRNA structure is shown in Figure 10.18 & 19. All tRNA have a similar 2nd/3rd structure, including: a Cloverleaf shaped structure plus 3 loops formed by hairpin turns; double helix regions [stabilized by Hydrogen bonding between bases] 58 Fig 10.18 Cloverleaf Structure of tRNA Molecules 59 10.3 RNA (3-D) Structural Elements tRNA Structure (cont.) The 3'-end (aka Acceptor Stem) contains the ester linkage for binding a specific amino acid. On the opposite end is the 3-base, Anticodon region, consisting of the triplet code complementary to mRNA [more later]. 60 10.3 RNA (3-D) Structural Elements Ribosomal RNA (rRNA) Structure rRNA structure has similarities to tRNA, except they are much larger. There are large regions of double helix structure. There are 3 types of Prokaryotic rRNA [5S, 16S, and 23S rRNA]. More in Chap 11; See Figure 10.20 61 End of Part I Chapter Seven ...
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