Chapter 10. Chapter 10 Nucleotides and Nucleic Acids. Nobel Prize Reginald H. Garrett Charles M. Grisham

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1 Reginald H. Garrett Charles M. Grisham Chapter 10 Nucleotides and Nucleic Acids Chapter 10 We have discovered the secret of life. Francis Crick, to patrons of The Eagle, a pub in Cambridge, England (1953) Nobel Prize 1962 Francis Crick (right) and James Watson (left) point out features of their model for the structure of DNA.

2 Information Transfer in Cells Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein See Figure 10.1 Information Transfer in Cells Figure 10.1 The fundamental process of information transfer in cells.

3 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Know the basic structures Pyrimidines Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA) Purines Adenine (DNA, RNA) Guanine (DNA, RNA) 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Figure 10.2 (a) The pyrimidine ring system; by convention, atoms are numbered as indicated. (b) The purine ring system; atoms numbered as shown.

4 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Figure 10.3 The common pyrimidine bases cytosine, uracil, and thymine 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Figure 10.4 The common purine bases adenine and guanine.

5 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Figure 10.5 Other naturally occurring purine derivatives hypoxanthine, xanthine, and uric acid. The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature The aromaticity and electron-rich nature of pyrimidines and purines enable them to undergo keto-enol tautomerism ( 互變異構性 ) The keto tautomers ( 互變異構物 ) of uracil, thymine, and guanine predominate at ph 7 By contrast, the enol form of cytosine predominates at ph 7 Protonation states of the nitrogens determines whether they can serve as H-bond donors or acceptors Aromaticity also accounts for strong absorption of UV light quantitative & qualitative analysis

6 The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature Figure 10.6 The keto-enol tautomerism of uracil. Figure 10.7 The tautomerization of the purine guanine. The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature Figure 10.8 The UV absorption spectra of the common ribonucleotides.

7 The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature Figure 10.8 The UV absorption spectra of the common ribonucleotides. Adenosine: A Nucleoside with Physiological Activity Adenosine functions as an autacoid ( 內分泌物 ), or local hormone, and neuromodulator ( 神經調節物質 ). Circulating in the bloodstream, it influences blood vessel dilation, smooth muscle contraction, neurotransmitter release, and fat metabolism. Adenosine is also a sleep regulator. Adenosine rises during extended wakefulness, promoting eventual sleepiness. Caffeine promotes wakefulness by blocking binding of adenosine to its neuronal receptors.

8 10.2 What Are Nucleosides? Structures to Know Nucleosides are compounds formed when a base is linked to a sugar via a glycosidic bond The sugars are pentoses D-ribose (in RNA) 2-deoxy-D-ribose (in DNA) The difference - 2'-OH vs 2'-H This difference affects secondary structure and stability 10.2 What Are Nucleosides? Figure 10.9 The linear and cyclic (furanose) forms of ribose.

9 10.2 What Are Nucleosides? Figure 10.9 The linear and cyclic (furanose) forms of deoxyribose What Are Nucleosides? The base is linked to the sugar via a glycosidic bond Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine Sugars make nucleosides more water-soluble than free bases

10 10.2 What Are Nucleosides? Uracil Adenine Cytosine Guanine Figure The common ribonucleosides What Is the Structure and Chemistry of Nucleotides? Nucleotides are nucleoside phosphates Know the nomenclature "Nucleotide phosphate" is redundant! wrong! Most monomeric ( 單體 ) nucleotides in the cells are ribonucleotides having 5 -phosphate groups (Fig ). Nucleoside diphosphates and triphosphates are nucleotides with two or three phosphate groups NDPs and NTPs are polyprotic acids ( 多質子酸 )

11 10.3 What Is the Structure and Chemistry of Nucleotides? Figure Structures of the four common ribonucleotides AMP, GMP, CMP, and UMP. Also shown: 3 -AMP What Is the Structure and Chemistry of Nucleotides? Cyclic nucleotides are cyclic phosphodiesters Figure The cyclic nucleotide camp.

12 10.3 What Is the Structure and Chemistry of Nucleotides? Figure The cyclic nucleotide cgmp 10.3 What Is the Structure and Chemistry of Nucleotides? Figure Formation of ADP and ATP by the successive addition of phosphate groups via phosphoric anhydride ( 磷酸酐 ) linkages. Note that the reaction is a dehydration synthesis.

13 10.3 What Is the Structure and Chemistry of Nucleotides? Figure Formation of ADP and ATP by the succesive addition of phosphate groups via phosphoric anhydride linkages. Note that the reaction is a dehydration synthesis. Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Nucleoside 5 -triphosphates are indispensable agents in metabolism because their phosphoric anhydride ( 磷酸酐 ) bonds are a source of chemical energy Bases serve as recognition units Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism

14 Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Figure Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Phosphoryl group transfer is shown here. Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Figure Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Pyrophosphoryl group transfer is shown here.

15 Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Figure Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Nucleotidyl group transfer is shown here What Are Nucleic Acids? Nucleic acids are linear polymers of nucleotides linked 3' to 5' by phosphodiester bridges Ribonucleic acid and deoxyribonucleic acid Sequence is always read 5' to 3' In terms of genetic information, this corresponds to "N to C" in proteins

16 10.4 What Are Nucleic Acids? Figure ',5'- phosphodiester bridges link nucleotides together to form polynucleotide chains. The 5'- ends of the chains are at the top; the 3'-ends are at the bottom What Are Nucleic Acids? Figure ,5 - phosphodiester bridges link nucleotides together to form polynucleotide chains. The 5 -ends of the chains are at the top; the 3 -ends are at the bottom.

17 10.5 What Are the Different Classes of Nucleic Acids? DNA - one type, one purpose RNA 3 or 4 types, 3 or 4 purposes ribosomal RNA - the basis of structure and function of ribosomes messenger RNA - carries the message for protein synthesis transfer RNA - carries the amino acids for protein synthesis small nuclear RNA: gene splicing Others: small RNAs: 21~28 nucleotides, gene regulation 10.5 What Are the Different Classes of Nucleic Acids? Figure The antiparallel nature of the DNA double helix.

18 The DNA Double Helix The double helix is stabilized by hydrogen bonds "Base pairs" arise from hydrogen bonds Erwin Chargaff had the pairing data, but didn't understand its implications Rosalind Franklin's X-ray diffraction data of DNA was crucial Francis Crick showed that it was a helix James Watson figured out the H bonds Chargaff s Data Held the Clue to Base Pairing

19 The Base Pairs Postulated by Watson Figure The Watson-Crick base pairs A:T and G:C. The Base Pairs Postulated by Watson Figure The Watson-Crick base pairs A:T and G:C.

20 The Structure of DNA Figure Replication of DNA gives identical progeny molecules because base pairing is the mechanism that determines the nucleotide sequence of each newly synthesized strand. The Structure of DNA An antiparallel double helix Diameter of 2 nm Length of 1.6 million nm (E. coli) Compact and folded (E. coli cell is only 2000 nm long) Eukaryotic DNA wrapped around histone ( 組織蛋白 ) proteins to form nucleosomes ( 核小體 ) Base pairs: A-T, G-C

21 Digestion of the E. coli cell wall releases the bacterial chromosome Figure The chromosome is shown surrounding the cell. Messenger RNA Carries the Sequence Information for Synthesis of a Protein Figure 10.20a Transcription and translation of mrna molecules in prokaryotic versus eukaryotic cells. In prokaryotes, a single mrna molecule may contain the information for the synthesis of several polypeptide chains within its nucleotide sequence.

22 Messenger RNA Carries the Sequence Information for Synthesis of a Protein heterogeneous nuclear RNA snrna+ proteins = snrnps Figure 10.20b Transcription and translation of mrna molecules in prokaryotic versus eukaryotic cells. In eukaryotes, a single mrna codes for just one protein, but structure is composed of introns and exons. Eukaryotic mrna DNA is transcribed to produce heterogeneous nuclear RNA (hnrna) mixed introns and exons with poly A intron - intervening sequence exon - coding sequence poly A tail 100~200 residues stability of mrna Splicing produces final mrna without introns

23 Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Ribosomes are about 2/3 RNA, 1/3 protein rrna serves as a scaffold for ribosomal proteins The different species of rrna are referred to according to their sedimentation coefficients rrnas typically contain certain modified nucleotides, including pseudouridine and ribothymidylic acid The role of ribosomes in biosynthesis of proteins is treated in detail in Chapter 30 Briefly: the genetic information in the nucleotide sequence of mrna is translated into the amino acid sequence of a polypeptide chain by ribosomes Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Figure Ribosomal RNA has a complex secondary structure due to many intrastrand H bonds. The gray line here traces a polynucleotide chain consisting of more than 1000 nucleotides. Aligned regions represent H- bonded complementary base sequences.

24 Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes S Figure The organization and composition of ribosomes. Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Figure Unusual bases in RNA.

25 Transfer RNAs Carry Amino Acids to Ribosomes for Use in Protein Synthesis Small polynucleotide chains - 73 to 94 residues each Several bases usually methylated Each a.a. has at least one unique trna which carries the a.a. to the ribosome 3'-terminal sequence is always CCA-a.a. Aminoacyl trna molecules are the substrates of protein synthesis Transfer RNAs Carry Amino Acids to Ribosomes for Use in Protein Synthesis Figure Transfer RNA also has a complex secondary structure due to many intrastrand hydrogen bonds. The black lines represent basepaired nucleotides in the sequence.

26 The RNA World and Early Evolution Thomas Cech and Sidney Altman showed that RNA molecules are not only informational they can also be catalytic This gave evidence to the postulate by Francis Crick and others that prebiotic evolution (that is, early evolution before cells arose) depended on self-replicating, catalytic RNAs But what was the origin of the nucleotides? A likely source may have been conversion of aminoimidazolecarbonitrile to adenine And glycolaldehyde could combine with other molecules to form ribose Adenine and glycolaldehyde exist in outer space The RNA World and Early Evolution Aminoimidazolecarbonitrile is tetramer of HCN and may be a precursor of adenine (a pentamer of HCN ).

27 The RNA World and Early Evolution Glycolaldehyde has been detected at the center of the Milky Way and could be a precursor of ribose and glucose. The Chemical Differences Between DNA and RNA Have Biological Significance Two fundamental chemical differences distinguish DNA from RNA: DNA contains 2-deoxyribose instead of ribose DNA contains thymine instead of uracil

28 The Chemical Differences Between DNA and RNA Have Biological Significance Why does DNA contain thymine? Cytosine spontaneously deaminates to form uracil Repair enzymes recognize these "mutations" and replace these Us with Cs But how would the repair enzymes distinguish natural U from mutant U Nature solves this dilemma by using thymine (5-methyl-U) in place of uracil The Chemical Differences Between DNA and RNA Have Biological Significance Figure Deamination of cytosine forms uracil.

29 The Chemical Differences Between DNA and RNA Have Biological Significance Figure The 5-methyl group on thymine labels it as a special kind of uracil. DNA & RNA Differences? Why is DNA 2'-deoxy and RNA is not? 2'-OH group in RNA make the 3'-phosphodiester bond of RNA more susceptible to alkaline hydrolysis DNA, lacking 2'-OH is more stable This makes sense - the genetic material must be more stable RNA is designed to be used and then broken down

30 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Figure Alkaline hydrolysis of RNA. Nucleophilic attach by OH - on the P atom leads to 5'-phosphoester cleavage Are Nucleic Acids Susceptible to Hydrolysis? Figure Alkaline hydrolysis of RNA. Nucleophilic attach by OH - on the P atom leads to 5'-phosphoester cleavage. Random hydrolysis of the cyclic phosphodiester intermediate gives a mixture of 2'- and 3'-nucleoside monophosphate products.

31 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Figure Alkaline hydrolysis of RNA. Random hydrolysis of the cyclic phosphodiester intermediate gives a mixture of 2'- and 3'-nucleoside monophosphate products Are Nucleic Acids Susceptible to Hydrolysis? RNA is resistant to dilute acid DNA is depurinated by dilute acid DNA is not susceptible to base RNA is hydrolyzed by dilute base

32 10.6 Are Nucleic Acids Susceptible to Hydrolysis? The Enzymes that hydrolyze nucleic acids are phosphodiesterases. Figure Cleavage in polynucleotide chains Are Nucleic Acids Susceptible to Hydrolysis? Figure 10.28ab Cleavage on the a side leaves the phosphate attached to the 5'-position of the adjacent nucleotide. b-side hydrolysis yields 3'-phosphate products.

33 Restriction Enzymes Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes" Type II and III restriction enzymes cleave DNA chains at selected sites Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage An enzyme that recognizes a 6-base sequence is a "six-cutter" Type II Restriction Enzymes No ATP requirement Recognition sites in dsdna have a 2-fold axis of symmetry Cleavage can leave staggered or "sticky" ends or can produce "blunt ends Names use 3-letter italicized code: 1st letter - genus; 2nd,3rd - species Following letter denotes strain EcoRI is the first restriction enzyme found in the R strain of E. coli

34 Cleavage Sequences of Restriction Endonucleases Restriction Mapping of DNA Figure Restriction mapping analysis.