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Chapter 8Nucleotides and Nucleic Acids

• Some basics

•Nucleic acid structure

•Nucleic acid chemistry

•Other functions of nucleotides

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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 mRNA into a specific sequence of amino acids.

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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 ：– 5C sugar （ ribose ）＋ N base ＋ Phosphate

• 二者間差異： – RNA ： ribose – DNA ： 2’-deoxyribose

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8.1 Some Basics1. 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 gr

oup.

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Structure of nucleotidesp. 274, Fig. 8-1a

DNA ：－ OH

RNA ：－ H

Purine ： N9

Pyrimidine ： N1

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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 gluc

osylated• 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

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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 pKa 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

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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

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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 a

t the right

pACGTA, pA-C-G-T-AOH, or pApCpGpTpA

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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 pH.

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Tautomeric forms of uracil. The lactam form predominates at pH 7.0; the other forms become more prominent as pH decreases. The other free pyrimidines and the free purines also have tautomeric forms, but they are more rarely encountered.

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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 pH of the cell.

• At acidic or alkaline pH 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.

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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 base-stacking interactions are very important in stabilizing the three-dimensional structure of nucleic acids.

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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 ani

mo groups

• H bonds btwn bases: – 2nd important mold of interaction btwn base

s– Permit complementary association of 2 stra

nds of nucleic acids– Base pairs ： A ＝ T(U) ； C≡G (p. 279, Fig. 8-

11)

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8.2 Nucleic Acid Structure

＊

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DNA stores genetic information–The Avery-MacLeod-McCarty experiment.

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The Hershey-Chase experiment

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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)

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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 – 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 36o；距離 3.4A ）– 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

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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.

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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. 8-18a)– Ribose 之 C1’ 與 nitrogen base 結合的鍵結可轉動 (p.284, Fig. 8-18b)

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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 )

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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 20o)

• Helix rise per base pair: 2.6A• 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]

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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

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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. 8-21, 迴文可能形成 hairpins or cruciforms)

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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

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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 pH, because the G≡C•C+ triplet requires a protonated cytosine. In the triplex, the pKa of this cytosine is > 7.5, altered from its normal value of 4.2.

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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.

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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. T

he purine strand (alternating A and G residues) is left unpaired. This structure produces a sharp bend in the DNA.

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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.

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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 could disrupt gene expression.

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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 mRNA molecule may code for one or several polypeptide chains.

• If it carries the code for only polypeptide, the mRNA is monocistronic; if it codes for two or more different polypeptides, the mRNA is polycistronic. (p. 288, Fig. 8-24)

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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.

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7. Many RNAs have more complex three-dimensional 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.

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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.

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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 pH and ionic strength and on the size and base composition of the DNA.

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Relationship between tm and the GC content of a DNA. (DNA 分子的熔點 )

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1. Double-helical DNA and RNA can be denatured

• Notably, RNA duplexes are more stable than DNA duplexes. At neutral pH, 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 pH, 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.

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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.

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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 GC 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 pH 3 causes selective removal of the purine bases, resulting in a derivative called apurinic acid.

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3. Nucleotides and nucleic acids undergo nonenzymatic transformations

• Formation of pyrimidine dimers induced by UV light. 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.

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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

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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 CpG 甲基化，產生對稱結構

• CpG 甲基化程度因 DNA 之區域而異• 甲基化可抑制 DNA 之 migration of seg

ments (transposons)

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p. 296, Fig. 8-35a Chemical agents that cause DNA

damage. Precursors of nitrous acid, which promotes deamination reaction.

亞硝酸鹽

硝酸鹽

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

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Chemical synthesis of DNA

DMT: an acid-labile dimethoxytrityl group

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Nucleotides carry chemical energy in cells

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The phosphate ester and phosphoanhydride bonds of ATP

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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.

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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-tRNA: （ p.148, Fig. 5.19 ）– AA at 3’ end of tRNA– anticodon loop– Procedures: p.149, Fig. 5.20

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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

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