Chapter 8 Nucleotides & Nucleic Acids

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1 Chapter 8 Nucleotides & Nucleic Acids We Need Nucleic Acids! RNA rrna DNA RNA mrna Protein Protein Trait Pol trna DNA contains genes, the information needed to synthesize functional proteins and RNAs DNA also contains segments that play a role in regulation of gene expression (promoters, operators, etc.) Messenger RNAs (mrnas) are transcribed from DNA by RNA polymerases and carry genetic information from a gene to the ribosome complex Ribosomal RNAs (rrnas) are components of the ribosomes and play a role in protein synthesis in conjunction with the template mrna and the AA carrying trna Transfer RNAs (trnas) carry the AAs designated by the codons of the mrna and bind to the Ribosome to help form the growing polypepide chain Chapter 8 2 1

2 Nucleotides and Nucleic Acids Nucleotides are the building blocks of nucleic acids A nitrogenous base A phosphate group (pyrimidines or purine) A pentose sugar Nucleotides have three characteristic components Chapter 8 3 Structure of a Nucleotide The Ribose Sugar Ribose (β-dribofuranose) is a pentose sugar 5 Note numbering of the carbons. In a nucleoside or nucleotide, "prime" is used (to differentiate from base numbering) Chapter 8 4 2

3 Structure of a Nucleotide The Pyrimidine and Purine Bases Big word, small ring Small word, big ring Know these: Numbering System and General Ring Structure Chapter 8 5 Structure of a Nucleotide Major Bases in Nucleic Acids The bases are abbreviated by their first letters (A, G, C, T, U). The purines (A, G) occur in both RNA and DNA The pyrimidine C occurs in both RNA and DNA, but T occurs only in DNA, and U occurs only in RNA Be able to recognize and draw these five! Chapter 8 6 3

4 Structure of a Nucleotide Bases from the minor leagues 5-Methylcytidine occurs in DNA of animals and higher plants N 6 -methyladenosine occurs in bacterial DNA These 8 bases you do not need to know Chapter 8 7 Structure of a Nucleotide The Phosphate A nucleoside + one or more phosphoryl groups is called a nucleotide. Chapter 8 8 4

5 Structure of a Nucleotide Linkages The phosphate at the 5 carbon Sugar to the base at 1 carbon Note the hydroxyl at the 2 carbon Why are these important? Chapter 8 9 Structure of a Nucleotide Nucleotides in Nucleic Acids Bases attach in β-linkage to the C-1' of ribose or deoxyribose The pyrimidines attach to the pentose via the N-1 position of the pyrimidine ring The purines attach through the N-9 position Some minor bases may have different attachments. Chapter

6 Structure of a Nucleotide Phosphate Variations: Number Nucleoside mono-, di-, or triphosphate Chapter 8 11 Structure of a Nucleotide Ribonucleotides The ribose sugar with a base (here, a pyrimidine, uracil or cytosine) attached to the ribose C-1' position is a ribonucleoside (here, uridine or cytidine). Phosphorylate the 5' position and you have a ribonucleotide (here, uridylate or cytidylate) Ribonucleotides are abbreviated (for example) U, or UMP (uridine monophosphate) Chapter

7 Structure of a Nucleotide The Major Ribonucleotides Note the N9 (purine) and N1 (pyrimidine) linkage to the ribose ring at C1 Chapter 8 13 Structure of a Nucleotide One of RNA s Little Problems Stability! DNA, which lacks a 2 -OH, is stable under alkaline conditions Chapter

8 Structure of a Nucleotide Deoxyribonucleotides Better for DNA! A 2'-deoxyribose sugar with a base (here, a purine - adenine or guanine) attached to the C- 1' position is a deoxyribonucleoside Phosphorylate the 5' position and you have a nucleotide Deoxyribonucleotides are abbreviated (for example) A, or da or damp Chapter 8 15 Structure of a Nucleotide The Major Deoxyribonucleotides Chapter

9 Structure of a Nucleotide Nucleotide Nomenclature Chapter 8 17 Structure of a Nucleotide Nucleotide Nomenclature Chapter

10 Structure of a Nucleotide Now, on to Nucleic Acids! Nucleotide monomers can be linked together via a phosphodiester linkage formed between the 3' -OH of a nucleotide and the phosphate of the next nucleotide. Two ends of the resulting poly- or oligonucleotide are defined: The 5' end lacks a nucleotide at the 5' position The 3' end lacks a nucleotide at the 3' end position Chapter 8 19 Structure of a Nucleotide Sugar-Phosphate Backbone Berg et al. Biochemistry 5 th Ed. Fig. 1.1 The polynucleotide or nucleic acid backbone thus consists of alternating phosphate and pentose residues. The bases are analogous to side chains of amino acids; they vary without changing the covalent backbone structure. Sequence is written from the 5' to 3' end: 5'-ATGCTAGC-3' Note that the backbone is polyanionic, and the phosphate groups have a pk a ~ 0. Chapter

11 Structure of a Nucleotide The bases can take syn- or anti- positions Chapter 8 21 Structure of a Nucleotide Sugar phosphate backbone conformation Polynucleotides have unrestricted rotation about most backbone bones (within limits of sterics) One exception is the sugar ring bond This behavior contrasts with the peptide backbone. Also in contrast with proteins, specific, predictable interactions between bases are often formed: A with T, and G with C. These interactions can be interstrand, or intrastrand. Chapter

12 Nucleic Acid Structure Polynucleotides vs. Polypeptides As in proteins, the sequence of side chains (bases in nucleic acids) plays an important role in function. Nucleic acid structure depends on the sequence of bases and on the type of ribose sugar (ribose, or 2'-deoxyribose). Hydrogen bonding interactions are especially important in nucleic acids, both interstrand and intrastrand. Chapter 8 23 Nucleic Acid Structure DNA DNA consists of two helical chains wound around the same axis in a right-handed fashion aligned in an antiparallel fashion. There are 10.5 base pairs, or 36 Å, per turn of the helix. Alternating deoxyribose and phosphate groups on the backbone form the outside of the helix. The planar purine and pyrimidine bases of both strands are stacked inside the helix. Chapter

13 Nucleic Acid Structure DNA The furanose ring usually is puckered in a C-2' endo conformation in DNA. The offset of the relationship of the base pairs to the strands gives a major and a minor groove. In B-form DNA (the most common) the depths of the major and minor grooves are similar to each other. The base-pairs are perpendicular to the helical axis Chapter 8 25 Nucleic Acid Structure DNA Strands The opposing strands of DNA are not identical, but are complementary. This means: they are positioned to align complementary base pairs: C with G, and A with T, but the strands run antiparallel to each other You can thus predict the sequence of one strand given the sequence of its complementary strand. Note that sequence conventionally is written from the 5' to 3' end Such a structure is useful for information storage and transfer! Chapter

14 Nucleic Acid Structure Stabilization of Double Helix Weak forces stabilize the double helix: (1) Hydrophobic Effects: Burying purine and pyrimidine rings in the interior of the helix excludes them from water (2) Stacking interactions: Stacked base pairs form van der Waals contacts (3) Hydrogen Bonds: H-bonding between the base pairs (not on the backbone!) (4) Charge-charge interactions: Electrostated repulsion of negatively charged phosphate groups is decreased by cations (e.g. Mg 2+ ) and cationic proteins Chapter 8 27 Nucleic Acid Structure Interstrand H-bonding Between DNA Bases Watson-Crick base pairing Chapter

15 Nucleic Acid Structure Base Stacking in DNA C-G (red) and A-T (blue) base pairs are isosteric (same shape and size), allowing stacking along a helical axis for any sequence. Base pairs stack inside the helix. Chapter 8 Berg et al. Biochemistry 5 th Ed. Figs. 1.4; Nucleic Acid Structure Various Nucleic Acid Structures B form - The most common conformation for DNA. A form - common for RNA because of different sugar pucker. Deeper minor groove, shallow major groove A form is favored in conditions of low water. Z form - narrow, deep minor groove. Major groove hardly existent. Can form for some DNA sequences; requires alternating syn and anti base configurations. 36 base pairs Backbone - blue; Bases- gray Chapter

16 Nucleic Acid Structure DNA Structure Summary Chapter 8 31 Nucleic Acid Structure Unusual DNA Structures Some structures seen in DNA are sequence dependent These structures can be very important in the binding of proteins to the nucleic acid Chapter

17 Nucleic Acid Structure RNA In gene expression, RNA acts as an intermediary between the genetic information of DNA and the production of polypeptides In eukaryotes, DNA is largely confined to the nucleus whereas protein production occurs in the cytoplasm. RNA allows these two processes to remain separated by shutting between them The product of transcription is always a single stranded RNA Tends to assume a right handed helical conformation dominated by base-stacking interactions Any self-complimentary sequences or interactions with a second RNA or a DNA chain will lead to duplex formation with one difference from duplex DNA: Uracil instead of Thymine Chapter 8 33 Nucleic Acid Structure RNA has a Rich and Varied Structure Watson-Crick base pairs in helical segments (usually A-form). Helix is secondary structure. Note A-U pairs in RNA DNA can also form structures like this. Chapter

18 Nucleic Acid Structure Large RNAs may be highly structured The 337 nt long M1 RNA of the trna processing enzyme RNase P of E. coli has many hairpin loops Even for a random RNA sequence, about 60-70% may be involved in secondary structure (why?) G-U pairs are also allowed, as they are energetically neutral. G-U pairs Other regions may also pair, which facilitates tertiary folding. Chapter 8 35 RNA Displays Interesting Tertiary Structure Singlestranded RNA right-handed helix Green = phosphodiester backbone Nucleic Acid Structure Yeast trna Phe (1TRA) Hammerhead ribozyme (1MME) T. thermophila intron, A ribozyme (RNA enzyme) (1GRZ) Chapter

19 Nucleic Acid Chemistry Replication: The preparation of exact copies of a molecule. DNA replication is essential for life, and is what allows organisms to create copies of themselves. Transcription: The copying of the genetic message encoded in DNA to RNA Translation: The translation of the genetic message encoded in RNA into a polypeptide with a specific amino acid sequence Performing these functions requires transformations of these complex molecules to occur without losing or damaging the genetic information (i.e. causing mutations). Thus, chemical modifications of nucleic acids must be prevented, controlled, monitored, and fixed if needed. Chapter 8 37 Nucleic Acid Chemistry Transformations of Nucleic Acids For DNA and RNA to perform their respective and various functions within organisms, they must undergo physical and chemical changes themselves Physical Denaturation (Melting) Renaturation (Annealing) Shearing Chemical Replication, synthesis Cleavage Deamination Depurination Crosslinking Alkylation Chapter 8 Oxidation 38 19

20 Nucleic Acid Chemistry DNA Replication In DNA replication, each DNA strand serves as a template for the synthesis of a new strand, producing two DNA molecules, each with one new strand and one old strand. This is termed semiconservative replication. Replication requires separation of the DNA strands (transient melting, or denaturation) so that the parent strands can serve as templates. Chapter 8 39 Nucleic Acid Chemistry Partially Denatured DNA DNA strands can be separated (DNA denaturation) by increasing temperature. Denaturation is a result of disruption of interactions that stabilize DNA structure: Hydrogen-bonding Base stacking This electron micrograph shows DNA that has been partially denatured with heat, and then fixed. The red arrows point to the single-stranded regions, which appear as bubbles in the doublestranded backbone. Would you expect these regions to be the same or different each time you denature this piece of DNA? Why or why not? What characteristics would these regions have? Chapter

21 Nucleic Acid Chemistry DNA Denaturation and T M Since DNA denaturation is commonly done by increasing temperature, it is also called DNA melting. This can be followed by observing the change in the ultraviolet absorption band of DNA. Stacked DNA bases absorb less light than unstacked; this is called hypochromism. The "melting temperature" T m is taken as the inflection point of the melting curve. Chapter 8 41 Nucleic Acid Chemistry DNA Melting and Annealing DNA denaturation is reversible. The formation of double helical DNA from denatured DNA is called annealing. Lowering the temperature below T m (given the right ph, ionic strength, etc.) will allow annealing. T m depends on ph, ionic strength, DNA length, and DNA base composition Chapter

22 Nucleic Acid Chemistry Annealing: Hybridization Experiment: completely denature duplex DNAs from a fly and a human. Mix the denatured samples and keep at ~ 65 C for many hours Under these conditions, complementary strands will anneal. Most fly strands anneal with fly; and human with human But some strands of fly and human DNA will anneal to give hybrid duplexes. This reflects a common evolutionary heritage. What would be different if this were done with human and Chapter 8 chimp DNA? 43 Nucleic Acid Chemistry Nucleic Acids Can Be Chemically Altered Deamination Depurination Crosslinking Alkylation Oxidation What will result from this? Chapter

23 Nucleic Acid Chemistry Base Deamination Nucleotide bases can undergo spontaneous loss of their exocyclic amino groups (deamination). Under typical cellular conditions, deamination of cytosine in DNA to uracil occurs in about one of every 10 7 residues every 24 hours. A and G deamination occurs at 1/10 of this rate. Can now pair with Chapter 8 45 Nucleic Acid Chemistry Mutation by Deamination Deamination of Cytosine is mutagenic. When deaminated, cytosine becomes uracil which pairs with A (rather than G) This mispairing causes a G to A substitution in the complementary strand. Uracil Chapter

24 Nucleic Acid Chemistry Mutation by Deamination Uracil Chapter 8 47 Nucleic Acid Chemistry Thymine in DNA Allows Error Correction! Why is U present in RNA, and T in DNA? Spontaneous deamination of U, which gives C, is relatively common. This is potentially mutagenic The enzyme uracil glycosylase recognizes U in DNA and cleaves the glycosidic bond to remove the base. Mutant cells lacking uracil glycosylase have a high rate of G-C to A-T base pair mutations. Chapter

25 Nucleic Acid Chemistry Depurination The N-β-glycosyl bond between the base and the pentose can undergo hydrolysis. This process is faster for purines than for pyrimidines 1/100,000 purines are lost from DNA every 24 hours Depurination of RNA and of ribonucleotides is much slower. Chapter 8 49 Nucleic Acid Chemistry Crosslinking of Bases Pyrimidine dimers can be formed by exposure to UV light. Most commonly seen for adjacent thymidine residues on the same DNA strand Cyclobutane, or 6-4-linked dimers, can be formed. This causes a kink in the helix X-rays and gamma rays can cause ring opening and base fragmentation. Chapter

26 Nucleic Acid Chemistry UV Damage to DNA Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA helix of these lesions occur in each skin cell for every second of sun exposure(!) Most of these are recognized and repaired immediately. If damage goes uncorrected, permanent mutation may result. CC to TT mutation occurs if a CC dimer is mispaired with two adenine bases. This is often seen in the p53 gene in skin cancers. Chapter 8 51 Nucleic Acid Chemistry Mutation by Methylation Methylation of the guanine carboxyl oxygen results in the formation of a nucleotide that can only form two hydrogen bonds with its partner This methylated gaunine can then be pair with T rather than C This is a weaker pair in addition to a mutagenic pairing Chapter

27 Nucleic Acid Chemistry Chemical Alkylating Agents Harmful to Children and Other Living Things Nitrogen mustard. First symptoms of exposure: skin, eye and respiratory tract irritation, burns and blistering. Long-term effects include leukemia. Used as a treatment of Hodgkin's disease and brain tumors (!) Chapter 8 53 Nucleic Acid Chemistry DNA Sequencing: Dideoxy Method (A) The Dideoxy method relies on the insertion of ddntps into a growing DNA chain, which causes chain termination. (B) Purified target DNA is synthesized using a primer specific for the strand to be sequenced. (C)In four separate reactions, four different ddntp s are added into the reaction mixture (in addition to the normal dntp s) resulting in the formation of DNA strands of varying lengths. (D)These strands can be separated using electrophoresis and the sequence read by comparing the lanes. Sequencing Animation Chapter

28 Nucleic Acid Chemistry Automated DNA Sequencing One major improvement in recent years has been the development of automated procedures for fluorescent DNA sequencing (Wilson et al., Genomics, 1990, pg. 626). These procedures generally use primers or dideoxynucleotides to which are attached fluorophores (chemical groups capable of fluorescing). During electrophoresis, a monitor detects and records the fluorescence signal as the DNA passes through a fixed point in the gel. The use of different fluorophores in the four base-specific reactions means that, unlike conventional DNA sequencing, all four reactions can be loaded into a single lane. The output is in the form of intensity profiles for each of the differently colored fluorophores, but the information is simultaneously stored electronically. Chapter 8 55 Figure 6.17 from Human Molecular Genetics, 2 nd Ed., Strachan et al. Nucleic Acid Chemistry Chemical Synthesis of DNA (A) NTP is attached to the silica support (B) DMT protecting group is removed (C)The incoming NTP is activated and coupled to the immobilized NTP to form a 5 to 3 linkage (D)The dinucleic acid is oxidized to produce the phosphotriester (E) Steps (B) through (D) are repeated (F) The remaining protecting groups are removed (G)The oligonucleotide is separated from the support Chapter 8 Why would synthesis of RNA be more difficult? 56 28

29 Other Functions of Nucleotides Building blocks of nucleic acids (RNA, DNA) (analogous to amino acid role in proteins) Energy currency in cellular metabolism (ATP: adenosine triphosphate) Allosteric effectors Structural components of many enzyme cofactors (NAD: nicotinamide adenine dinucleotide) Chapter