Chapter 8 Nucleotides and Nucleic Acids Some basics

Chapter 8 Nucleotides and Nucleic Acids • Some basics • Nucleic acid structure • Nucleic acid chemistry • Other functions of nucleotides Oct, 2007 1

8. 1 Some Basics • Gene–A segment of a DNA molecule that contains the information required for the synthesis of a functional biological product. • RNAs– 1. Ribosomal RNAs –structural components of ribosomes, the complexes that carry out the synthesis of proteins. 2. Messenger RNAs– intermediaries, carrying genetic information from one or a few genes to a ribosome, where the corresponding proteins can be synthesized. 3. Transfer RNAs– adapter molecules that faithfully translate the information in m. RNA into a specific sequence of amino acids. Oct, 2007 2

8. 1 Some Basics • Two types of nucleic acids：DNA & RNA – RNA：Ribonucleic acid – DNA：Deoxyribonucleic acid （p. 85） • 二者共同特色：均為polymers – Monomer unit： – 5 C sugar（ribose）＋N base＋Phosphate • 二者間差異： – RNA：ribose – DNA： 2’-deoxyribose Oct, 2007 3

8. 1 Some Basics 1. Nucleotides and Nucleic Acids Have Characteristic Bases and Pentoses • Nucleotides – have three characteristic components: (1) A nitrogenous base: purine (adenine (A) and guanine (G)) pyrimidine： (cytosine (C), thymine (T) [for DNA] and uracil (U) [for RNA] ) (2) A pentose (3) A phosphate • Nucleoside – A nucleotide without the phosphate group. Oct, 2007 4

Purine：N 9 Pyrimidine：N 1 DNA：－OH RNA：－H Structure of nucleotides Oct, 2007 p. 274, Fig. 8 -1 a 5

1. Nucleotides and Nucleic Acids Have Characteristic Bases and Pentoses • • P. 276, Fig. 8 -5 a&b 較少見的purine and pyrimidine bases DNA：methylated forms Viral DNAs：hydroxymethylated or glucosylated • 角色：調控或保護遺傳訊息 • 命名原則：替代的官能基所在的環的位置 • P. 276, Fig. 8 -6：磷酸化的核苷 Oct, 2007 6

2. Phosphodiester Bonds Link Successive Nucleotides in Nucleic Acids • Nucleotides 以phosphodiester linkage 結合形成 核酸 (P. 277, Fig. 8 -7) • The Nature & Significance of Primary Structure – 方向性：因phosphodiester link之連接方式，nucleic acids有3’-end，5’-end之分別 – 獨特性：組成核酸分子之nucleotide sequence • 核酸骨幹由磷酸根與核糖交錯串成，具高度親水 性 • 磷酸之p. Ka近乎0，生理環境下帶負電荷，藉周圍 帶正電荷之蛋白質、金屬離子、多胺 (polyamines)中和其負電荷 Oct, 2007 7

2. Phosphodiester Bonds Link Successive Nucleotides in Nucleic Acids • 5 -end與3‘-end之定義 – The 5’ end lacks a nucleotide at the 5’ position – The 3’ end lacks a nucleotide at the 3’ position. • RNA is hydrolyzed rapidly under alkaline conditions, but DNA is not; the 2’-hydroxyl group in RNA are directly involved in the process. (RNA在鹼性環境下迅速水解，與2’ －OH有關，p. 277, Fig. 8 -8) • Oligonucleotide：≤ 50 nucleotides • Polynucleotide：＞ 50 nucleotides Oct, 2007 8

核酸結構的簡要表達方式 By convention, the structure of a single strand of nucleic acid is always written with the 5’ end at the left and 3’ end at the right p. ACGTA, Oct, 2007 p. A-C-G-T-AOH, or p. Ap. Cp. Gp. Tp. A 9

3. The Properties of Nucleotide Bases Affect the Three-Dimentional Structure of Nucleic Acids • Free pyrimidines and purines are weakly basic compounds and thus called bases. • Resonance among atoms in the ring gives most of the bonds partial double-bond character. One result is that pyrimidines are planar molecules; purines are very nearly planar, with slight pucker. • Free pyrimidine and purine bases may exist in two or more tautomeric forms depending on the p. H. Oct, 2007 10

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Tautomeric forms of uracil. The lactam form predominates at p. H 7. 0; the other forms become more prominent as p. H decreases. The other free pyrimidines and the free purines also have tautomeric forms, but they are more rarely encountered. Oct, 2007 12

3. The Properties of Nucleotide Bases Affect the Three-Dimentional Structure of Nucleic Acids • The purine and pyrimidine bases are hydrophobic and relatively insoluble in water at the near-neutral p. H of the cell. • At acidic or alkaline p. H the bases become charged and their solubility in water increases. • Hydrophobic stacking interactions in which two or more bases are positioned with the planes of their rings parallel are one of two important modes of interaction between bases in nucleic acids. Oct, 2007 13

3. The Properties of Nucleotide Bases Affect the Three-Dimentional Structure of Nucleic Acids • The stacking also involved a combination of van der Waals and dipole-dipole interactions between bases. • Base stacking helps to minimize contact of the bases with water, and basestacking interactions are very important in stabilizing the three-dimensional structure of nucleic acids. Oct, 2007 14

3. The Properties of Nucleotide Bases Affect the Three-Dimentional Structure of Nucleic Acids • Purine & pyrimidine最重要的官能基： – Ring N、carbonyl groups、exocyclic animo groups • H bonds btwn bases: – 2 nd important mold of interaction btwn bases – Permit complementary association of 2 strands of nucleic acids – Base pairs：A＝T(U) ；C≡G (p. 279, Fig. 8 -11) Oct, 2007 15

8. 2 Nucleic Acid Structure Oct, 2007 ＊ 16

DNA stores genetic information–The Avery-Mac. Leod-Mc. Carty experiment. Oct, 2007 17

The Hershey-Chase experiment Oct, 2007 18

2. DNA Molecules Have Distinctive Base Compositions • The base composition of DNA generally varies from one species to another. • DNA specimens isolated from different tissues of the same species have the same base composition. • The base composition of DNA in a given species does not change with an organism’s age, nutritional state, or changing enviroment. • A =T, and G =C. Thus, A + G = T + C. (Chargaff’s rules) Oct, 2007 19

3. DNA Is a Double Helix • Watson & Crick model (1953)說明DNA之二級結構 (Nobel Prize, 1962) • Franklin & Wilkins提供X-ray diffraction證據 (p. 282, Fig 8 -14) – – – – 螺旋體結構，2 DNA strands上的鹼基以氫鍵結合 鹼基配對（base pairing）：A＝T；C≡G 10 pairs/turn （base pairs間彼此相錯 36 o；距離 3. 4 A） Height： 3. 4 nm/turn (34 A/turn) Antiparallel（反向性質）(p. 283, Fig. 8 -16) 2 DNA strands間之互補（complementary）關係 Self-replication：Chargaff’s rules：A＋G＝C＋T Oct, 2007 20

4. DNA Can Occur in Different Three. Dimentional Forms (1) Structural variation generally do not affect the key properties of DNA defined by Watson and Crick: strand complementarity, antiparallel strands, and requirement for A=T and G≡C base pairs. Oct, 2007 21

4. DNA Can Occur in Different Three. Dimentional Forms • (2) DNA不同的結構反映 3個狀況 – Deoxyribose可能有不同的conformations – 核酸骨幹構成鍵結可轉動 (p. 284, Fig. 8 -18 a) – Ribose之C 1’與nitrogen base結合的鍵結可轉動 (p. 284, Fig. 8 -18 b) Oct, 2007 22

4. DNA Can Occur in Different Three. Dimentional Forms • Alternative nucleic acid structures: B, A, Z helices – B form • 存在於高溼度狀態 • Helix rise per base pair: 3. 4 A • Base pairs與中心軸較近 (DNA strands橫切面 直徑較小) • DNA strands表面有大小不同的溝隙 (major & minor grooves) • 右旋結構 • 1 turn中含 10. 5 bp (p. 284, Fig. 8 -19, Table ) Oct, 2007 23

4. DNA Can Occur in Different Three. Dimentional Forms – A form • 存在於低濕度狀態(脫水、結晶) 例：double-strand RNA、DNA-RNA hybrid • Base pairs離中心軸較遠 (double helix橫切面直徑較B form大) • Base pairs與中心軸非垂直(傾斜角度大, 約20 o) • Helix rise per base pair: 2. 6 A • Double helix表面溝紋均勻 • 缺水狀況下穩定 • 右旋結構 • 1 turn含 11 bp [p. 284, Fig. 8 -19] Oct, 2007 24

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4. DNA Can Occur in Different Three. Dimentional Forms • Z form – 左旋 – 12 bp/turn, slender, more elongated – Zigzag backbone – Purine-pyrimidine序列特色: • Alternating C, G • Alternating 5 -methyl C, G • Alternating syn-purine, anti-pyrimidine – 可能具有調控特定基因表現的功能 Oct, 2007 26

5. Certain DNA sequences adopt unusual structures • Bends occur in the DNA helix whenever four or more adenosine residues appear sequentially in one strand. The bending sequences of DNA provide the sites for the binding by the special proteins. (-AAAA-) • Hairpins–Palindromic DNA sequences in the same strand. (p. 285, Fig. 8 -20, 迴文) • Cruciforms–Palindromic DNA sequences in both strands of a duplex DNA. (p. 285, Fig. 821, 迴文可能形成hairpins or cruciforms) Oct, 2007 27

Triplex DNAs Base-pairing patterns in one well-characterized form of triplex DNA. The Hoogsteen pair in each case is shown in red. 結構不尋常, 可能是DNA代謝啟動或調控處 Oct, 2007 28

Hoogsteen Pairing • A cytidine residue (if protonated) can pair with the guanosine residue of a G≡C nucleotide pair, and a thymidine can pair with the adenosine of an A=T pair. • Hoogsteen pairing allows the formation of triplex DNAs that are stable at the low p. H, because the G≡C • C+ triplet requires a protonated cytosine. In the triplex, the p. Ka of this cytosine is > 7. 5, altered from its normal value of 4. 2. Oct, 2007 29

G Tetraplex • Four DNA strands can also pair to form a tetraplex (also called quadruplex), but this occurs readily only for DNA sequences with a very high proportion of guanosine residues. • The orientation of strands in the tetraplex can be parallel or antiparallel. Oct, 2007 30

H-DNA: 出現在可辨識特定序列的DNA-Bining Proteins的結合處, 與基因調控有關 A sequence of alternating T and C residues can be considered a mirror repeat centered about a central T or C. The purine strand (alternating A and G residues) is left unpaired. This structure produces a sharp bend in the DNA. Oct, 2007 31

These sequences from an unusual structure in which the strands in one half of the mirror repeat are separated and the pyrimidine-containing strand (alternating T and C residues) folds back on the other half of the repeat to form a triplex helix. Oct, 2007 32

5. Certain DNA sequences adopt unusual structures • In the DNA of living cells, sites recognized by many sequence-specific DNA-binding proteins are arranged as palindromes, and polypyrimidine or polypurine sequences that can form triplex helices or even H-DNA are found within regions involved in the regulation of expression of some eukaryotic genes. • Synthetic DNA strands designed to pair with the regulatory sequences of DNA to form triplex DNA could disrupt gene expression. Oct, 2007 33

6. Messenger RNAs code for polypeptide chains • RNA acts as an intermediary by using the information encoded in DNA to specify the amino acid sequence of a function protein. • In prokaryotes a single m. RNA molecule may code for one or several polypeptide chains. • If it carries the code for only polypeptide, the m. RNA is monocistronic; if it codes for two or more different polypeptides, the m. RNA is polycistronic. (p. 288, Fig. 8 -24) Oct, 2007 34

7. Many RNAs have more complex three-dimensional structures • RNA can base-pair with complementary regions of either RNA or DNA. • Base pairing matches the pattern for DNA: G pairs with C and A pairs with U. One difference is that base pairing between G and U residues–unusual in DNA–is fairly common in RNA. • The paired strands in RNA or RNA-DNA duplexes are antiparallel, as in DNA. Oct, 2007 35

7. Many RNAs have more complex threedimensional structures • When complementary sequences are present, the predominant double-stranded structure is an A-form right-handed double helix. Z-form helices have been made in the laboratory. The B form of RNA has not been observed. • Breaks in the regular A-form helix caused by mismatched or unmatched bases in one or both strands are common and result in bulges or internal loops. • Specific short base sequences (e. g. UUCG) are often found at the ends of RNA hairpins and are known to form particular tight and stable loops. Oct, 2007 36

1. Double-helical DNA and RNA can be denatured • The close interaction between stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same concentration of free nucleotides. • The absorption is decreased further when two complementary nucleic acids are paired. This is called the hypochromic effect. • Denaturation of a double-stranded nucleic acid produces the opposite result, an increase in absorption called the hyperchromic effect. • The transition from double-stranded DNA to the single-stranded, denatured form can thus be detected by monitoring the absorption of UV light. Oct, 2007 37

Oct, 2007 Heat denaturation of DNA. The denaturation of melting curves of two DNA specimens. The temperature at the midpoint of the transition (tm) is the melting point. It depends on p. H and ionic strength and on the size and base 38 composition of the DNA.

Oct, 2007 Relationship between tm and the G C content of a DNA. (DNA分子的熔點) 39

1. Double-helical DNA and RNA can be denatured • Notably, RNA duplexes are more stable than DNA duplexes. At neutral p. H, denaturation of a double-helical RNA often requires temperatures 20°C or more higher than those required for denaturation of a DNA molecule with a comparable sequence. (在中性p. H, 雙股 RNA較雙股DNA穩定，熔點較高) • The stability of an RNA-DNA hybrid is generally intermediate between that of RNA and that of DNA. Oct, 2007 40

2. Nucleic Acids from Different Species Can Form Hybrids p. 293, Fig. 8 -32 DNA hybridization Two DNA samples to be compared are completely denatured by heating. When the two solutions are mixed and slowly cooled, DNA strands of each sample associated with their normal complementary partner and anneal to form duplexes. If the two DNAs have significant sequence similarity, they also tend to form partial duplexes or hybrids with each other: the greater the sequence similarity of hybrids formed. Oct, 2007 41

3. Nucleotides and nucleic acids undergo nonenzymatic transformations • Deamination reactions: deamination of cytosine (in DNA) to uracil occurs in about one every 107 cytidine residues in 24 hours. Deamination of adenine and guanine is about 100 times slower. Cytosine deamination would gradually lead to a decrease in G C base pairs and an increase in A=U base pairs in the DNA of all cells. • Depurination: As many as one in 105 purines are lost from DNA every 24 hours under typical cellular conditions. Incubation of DNA at p. H 3 causes selective removal of the purine bases, resulting in a derivative called apurinic acid. Oct, 2007 42

3. Nucleotides and nucleic acids undergo nonenzymatic transformations • Formation of pyrimidine dimers induced by UV light induces the condensation of two ethylene groups to for a cyclobutane ring or form a 6 -4 photoproduct. • Both x rays and gamma rays can cause ring opening and fragmentation of bases as well as breaks in the covalent backbone of nucleic acids. • Chemical reagents that cause DNA damage. • Oxidative damages: Excited-oxygen species such as hydrogen peroxide, hydroxyl radicals, and superoxide radicals arise during irradiation or as a byproduct of aerobic metabolism. Oct, 2007 43

4. Some Bases of DNA Are Methylated • DNA特定區域或序列的特定鹼基會被甲基化； A、C較G、T頻繁 • All DNA methylase用S-adenosylmethionine 為methyl group donor • Methylation之功能？ • E. coli有二甲基化系統 – 辨識本身（甲基化）與外來（未甲基化）DNA (restriction-modification system) – Dam (DNA adenine methylation) methylase將 (5’)GATC(3’)之A為 6‘-methyladenosine：修復 mismatched BP Oct, 2007 44

4. Some Bases of DNA Are Methylated • Eukryotic cells：DNA約5%之C被甲基化 為 5 -methylcytidine • 主要將DNA雙股之Cp. G甲基化，產生對 稱結構 • Cp. G甲基化程度因DNA之區域而異 • 甲基化可抑制DNA 之migration of segments (transposons) Oct, 2007 45

亞硝酸鹽 p. 296, Fig. 8 -35 a Chemical agents that cause DNA damage. Precursors of nitrous acid, which promotes deamination reaction. Oct, 2007 46

4. The DNA Sequences of Long DNA Strands Can Be Determined: DNA sequencing by the Sanger method Oct, 2007 47

Chemical synthesis of DNA DMT: an acid-labile dimethoxytrityl group Oct, 2007 48

Nucleotides carry chemical energy in cells Oct, 2007 49

The phosphate ester and phosphoanhydride bonds of ATP Oct, 2007 50

The UV absorption spectra of NAD+ and NADH. Reduction of the nicotinamide ring produces a new, broad absorption band with a maximum at 340 nm. Oct, 2007 51

四� From Gene to Protein • The Genetic Code: （p. 147, Fig. 5. 16） – Start signal: AUG • Prokaryotes: N-formylmethionine • Eukaryotes: methionine at N-terminus – Stop signals: UAA, UAG, UGA • Translation: （p. 147, Fig. 5. 18） – aminoacyl-t. RNA: （p. 148, Fig. 5. 19） – AA at 3’ end of t. RNA – anticodon loop – Procedures: p. 149, Fig. 5. 20 Oct, 2007 52

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四� From Gene to Protein • Posttranslational Processing of Proteins –[以insulin為例 (p. 149, Fig. 5. 21)] –在 Protein 生成後，所進行之進一步的結 構修飾作用，以形成在生物細胞中具有完整 結構與功能的protein之過程，稱之 Oct, 2007 58

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