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Chapter 28 Biomolecules: Heterocycles and Nucleic Acids Heterocycles: cyclic organic compounds that contain rings atoms other than carbon (N,S,O are the most common). 28.1 28.2 Five-Membered Unsaturated Heterocycles (please read) Structures of Pyrrole, Furan, and Thiophene (please read) 3 3 N1 H Pyrole 28.3 28.4 28.5 28.6 28.7 3 2 2 O1 Furan 2 S1 Thiophene Cyclopentadienyl anion Electrophilic Substitution Reactions of Pyrrole, Furan, and Thiophene (please read) Pyridine, a Six-Membered Heterocycle (please read) Electrophilic Substitution of Pyridine (please read) Nucleophilic Substitution of Pyridine (please read) Fused-Ring Heterocycles (please read) These sections contain some important concepts that were 418 covered previously. 28.8 Nucleic Acids and Nucleotides Nucleic acids are the third class of biopolymers (polysaccharides and proteins being the others) Two major classes of nucleic acids deoxyribonucleic acid (DNA): carrier of genetic information ribonucleic acid (RNA): an intermediate in the expression of genetic information and other diverse roles The Central Dogma (F. Crick): DNA (genome) mRNA Protein (proteome) The monomeric units for nucleic acids are nucleotides Nucleotides are made up of three structural subunits 1. Sugar: ribose in RNA, 2-deoxyribose in DNA 2. Heterocyclic base 3. Phosphate 419 212 Nucleoside, nucleotides and nucleic acids phosphate base sugar phosphate phosphate base sugar base sugar base sugar phosphate nucleoside nucleotides base sugar nucleic acids The chemical linkage between monomer units in nucleic acids 420 is a phosphodiester The sugar: based on the furanose form D-ribose ribose for RNA; 2-deoxyribose for DNA CHO H OH H OH HO 5 OH 3 CH2OH HO H HO O 1 OH 4 O OH 2 OH HO D-ribose D-2-deoxyribose The heterocyclic base; there are five common bases for nucleic acids 7 N 5 N H 4 NH2 6 N 8 9 N3 1 N 2 N H N H NH2 4 6 N N adenine (A) DNA/RNA purine 5 N 3 2 N1 pyrimidine O N O N H O cytosine (C) DNA/RNA NH2 guanine (G) DNA/RNA H3C N NH N O NH N H O thymine (T) DNA NH N H O uracil (U) RNA 421 213 Nucleosides: sugar + base ribonucleosides or 2’-deoxyribonucleosides NH2 7 5 N 8 5' HO O 6 N 9 N 4 N N 2 HO 3 1' 4' O 1 N NH2 2' 3' X HO RNA: X= OH, guanosine (G) DNA: X= H, 2'-deoxyguanosine (dG) NH2 N O HO X HO O O H3C N O X HO RNA: X= OH, adenosine (A) DNA: X= H, 2'-deoxyadenosine (dA) HO O NH N NH NH O N O HO O DNA: thymidine (T) O OH HO HO RNA: X= OH, cytidine (C) DNA: X= H, 2'-deoxycytidine (dC) N RNA: R= H, uridine (U) 422 Nucleotides: nucleoside + phosphate HO HO O HO P O O B O HO X ribonucleoside (X=OH) deoxyribonucleoside (X=H) O O HO P O P O O O B O HO X nucleotide 5'-diphosphate 28.9 B O X ribonucleotide 5'-monophosphate (X=OH) deoxyribonucleotide 5'-monophosphate (X=H) O O O HO P O P O P O O O O B O O B O O HO P O X nucleotide 5'-triphosphate Structure of nucleic acids: The chemical linkage between nucleotide units in nucleic acids is a phosphodiester, which connects the 5’-hydroxyl group of one nucleotide to the 3’-hydroxyl group of the next. O OH ribonucleotide 3',5'-cyclic phosphosphate 3' O O P O O 5' Base O 3' O X O P O O 5' O Base 3' DNA is negatively charged O 5' X 423 214 DNA sequences are written from left to right from the 5’ to 3’ 5’-ATCGCAT-3’ or 5’-d(ATCGCAT)-3’ 28.10 Base Pairing in DNA: The Watson–Crick Model Chargaff’s Rule: the number of A = T and G = C in DNA Two polynucleotide strands, running in opposite directions (anti-parallel) and coiled around each other in a double helix. The strands are held together by complementary hydrogenbonding between specific pairs of bases. “Molecular Structure of Nucleic Acids” Watson J. D.; Crick, F. H. C. Nature 1953, 171, 737-738 "Molecular Structure of Deoxypentose Nucleic Acids" Wilkins, M. H. F.; Stokes A.R.; Wilson, H. R. Nature 1953,171, 738-740. ”Molecular Configuration in Sodium Thymonucleate,” Franklin, R.; Gosling, R. G. Nature 1953, 171, 740-741 1962 Nobel Prize in Medicine: F. H. C. Crick, J. D. Watson, Maurice F. H. Wilkins, “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material.” 424 Anti-parallel, complementary hydrogen-bonding of DNA base pairs Antiparallel C-G Pair Hydrogen Bond Donor N 5' O2 H O N 5' Hydrogen Bond N3 Acceptor Hydrogen Bond Acceptor Bond O6 Hydrogen Acceptor H N4 RO N H N O H N N N N O N1 Hydrogen Bond RO Donor O Hydrogen Bond N2 Donor H OR 3' OR 3' Antiparallel T-A Pair Hydrogen Bond Acceptor H O4 Hydrogen Bond N3 Donor O 5' RO N H OR N6 N Hydrogen Bond Donor 5' N N N N O H N RO O N1 Hydrogen Bond Acceptor O OR 3' 3' 425 215 DNA double helix major groove 12 Å one helical turn 34 Å minor groove 6Å backbone: deoxyribose and phosphodiester linkage bases 426 DNA Grooves major groove guanosine cytidine minor groove major groove adenosine thymidine minor groove 427 216 “It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick 28.11 Nucleic Acids and Heredity The Central Dogma (F. Crick): DNA DNA transcription mRNA (genome) replication translation Protein (proteome) Expression and transfer of genetic information: Replication: process by which DNA is copied with very high fidelity. Transcription: process by which the DNA genetic code is read and transferred to messenger RNA (mRNA). This is an intermediate step in protein expression Translation: The process by which the genetic code is converted to a protein, the end product of gene expression. The DNA sequence codes for the mRNA sequence, which 428 codes for the protein sequence 28.12 Replication of DNA: DNA is replicated by the coordinated efforts of a number of proteins and enzymes. Each cell contains about two meters of DNA. The DNA must be “packaged” into the cell nucleus by super-coiling and knotting. For replication, DNA must be unknotted, uncoiled and the double helix unwound. Topoisomerase: Enzyme that unknots and uncoils DNA Helicase: Protein that unwinds the DNA double helix. DNA polymerase: Enzyme replicates DNA using each strand as a template for the newly synthesized strand. DNA replication is semi-conservative: Each new strand of DNA contains one parental (old, template) strand and one daughter (newly synthesized) strand 429 217 Unwinding of DNA by helicases expose the DNA bases (replication fork) so that replication can take place leading strand DNA polymerase δ DNA replication Lagging strand DNA polymerase ε 430 DNA Polymerase: the new strand is replicated from the 5’ → 3’ (start from the 3’-end of the template) DNA polymerases are Mg2+ ion dependent The deoxynucleotide 5’-triphosphate (dNTP) is the reagent for nucleotide incorporation OH 5' O O O O P O P O P OO O- O- O T A O O O O P -O O template strand (old) Mg2+ OH O G O dNTP O C 5' (new) O 3' 3’-hydroxyl group of the growing DNA strand acts as a nucleophile and attacks the α-phosphorus atom of the dNTP. 431 218 Replication of the leading strand occurs continuously in the 5’ → 3’ direction of the new strand. Replication of the lagging strand occurs discontinuously. Short DNA fragments are initially synthesized and then ligated together. DNA ligase catalyzes the formation of the phosphodiester bond between pieces of DNA. animations of DNA processing: http://www.wehi.edu.au/education/wehi-tv/dna/ 432 DNA replication occurs with very high fidelity: Most DNA polymerases have high intrinsic fidelity Many DNA polymerases have “proof-reading” (exonuclease) activity Mismatch repair proteins seek out and repair base-pair mismatches due to unfaithful replication 28.13 Structure and Synthesis of RNA: Transcription RNA contains ribose rather than 2-deoxyribose and uracil rather than thymine There are three major kinds of RNA messenger RNA (mRNA): ribosomal RNA (rRNA) transfer RNA (tRNA) DNA is found in the cell nucleus and mitochondria; RNA is more 433 disperse in the cell. 219 Transcription: only one of the DNA strands is copied (coding or antisense strand). It sequence is converted to the complementary sequence in mRNA (template or sense strand), which codes for the amino acid sequence of a protein (or peptide) 28.14 RNA and Protein Biosynthesis: Translation • proteins are synthesized in the cytoplasm on ribosomes. • mRNA is the template for protein biosynthesis. • a three base segment of mRNA (codon) codes for a an amino acid. 434 The “anticodon” region of of tRNA is complementary for a to the mRNA codon sequence. The t-RNA carries an amino acid on the 3’-hydroxyl (aminoacyl t-RNA) and the ribosome catalyzes amide bond formation. Although single-stranded, the are complementary sequences within tRNA that give it a defined conformation aminoacyl t-RNA 435 220 Ribosomal protein synthesis OH H3CS H3CS H2N H2N H2N O O O O U A C U A C A U G U A U G C U C O A U A A U G U A U G C U C U U U U 3' 5' 3' 5' H3CS H3CS O H2N OH O U A C O H2N HN HO O OH H2N HN O HO A U A A U G U A U G C U C U A C U U 5' O O O A U A C G A CH3 O A U G U A U G C U C U U 3' 3' 5' 3' 436 DNA sequencing Maxam-Gilbert: relys on reagents that react with a specific DNA base that can subsequent give rise to a sequence specific cleavage of DNA Sanger: Enzymatic replication of the DNA fragment to be sequenced with DNA polymerase, Mg+2, and dideoxynucleotides triphosphate (ddNTP) that truncate DNA replication Restriction endonucleases: Bacterial enzymes that cleave DNA at specific sequences 5’-d(G-A-A-T-T-C)-3’ 3’-d(C-T-T-A-A-G)-5’ 5’-d(G-G-A-T-C-C)-3’ 3’-d(C-C-T-A-G-G)-5’ 5' 3' 3' 5' EcoR I restriction enzyme 2-O PO 3 OH 3' 5' BAM HI 5' 3' 2-O PO 3 OH 437 221 Sanger Sequencing dideoxynucleotides triphosphate (ddNTP) NH2 N O -O O O N P O P O P O O- O- -O O O O P O P O P O O- O- O- ddATP 3' N NH O N N NH2 O O N O O O -O O N P O P O P O O- O- O O- N NH N O NH2 -O O O O- O O O- ddGTP ddTTP N P O P O P O O- O- O ddCTP 5'-32P primer Anti-Viral Nucleosides When a ddNTP is incorporated elongation of the primer is terminated 3' DNA polymerase O HO O O NH N N N3 AZT The ddNTP is specifically incorporated opposite its complementary nucleotide base Mg2+, dNTP's + one ddATP N O O N NH HO ddI NH2 HO O H2N N N O O N N OH O S (-)-3TC d4C 5' Template unknown sequence 438 Sanger Sequencing ddA ddG ddC ddT 5' Larger fragments Smaller fragments C A T T G C A T T A G T G T C G T A A C G T A A T C A C A G 5' 3' 32P 3' 32P-5' primer 3' template 439 222 Automated Sanger Sequencing with flourescent ddNTP's H N O HN O -3 H3C N O H3O9P3O O N N O -3 (CH2)2 H3C CH3 N O N CH3 O N CH3 HN CH3 NH2 N N H3O9P3O O O O NH O- N -3 O O- O O CH3 N H3O9P3O N NH2 O CH3 ddA ddC Excitation: ~ 490 nM, O O- O O -3 ddT O N H3C (CH2)2 O O- O O CH3 HN H3C H3O9P3O CH3 O O H N NH2 O ddG Emission: ddT= 526 nm. ddC= 519 nm, ddA= 512 nm, ddG= 505 nm Sanger sequencing using flourescent ddNTP's: terminated DNA strands are separated by capillary electrophoresis, detected and identified by their fluorescence emission 440 Sequencing on a “chip” (Lagniappe) Spatially addressable: 8-mer chip: 65, 536 different sequences 12-mer chip: 1,677,216 different sequences DNA fragment DNA–fluorophore Place DNA on the chip, then wash away non-specific hybridization after 1-10 hrs. Raise temperature and “melt” away partially hybridized sequences. 3’-ACGGTGCG CGGTGCGA GGTGCGAG GTGCGAGA TGCGAGAA GCGAGAAT etc 3’-ACGGTGCGAGAAT---5’ 5’-TGCCACGCTCTTA---3’ (from the chip) 441 223 28.16 DNA Synthesis: short segments of DNA can be efficiently by automated, solid-phase methods. Solid support: controlled pore glass (silica) linked to the 3’-hydroxyl group of the first nucleotide solid phase DNA synthesis is from 3’→5’, which is the opposite direction from nature. Protected nucleotide reagents: 5’-protecting group: 4,4’-dimethyoxytrityl (trityl is a triphenylmethyl group), abbreviated DMT OCH3 5’-DMT ether C O O Removes with mild acid (Cl3CCO2H) Base HO 442 OCH3 The base amino groups of dA, dG and dC must be protected, usually as an amides. The base of T does not require further protection. O O HN N N N HN O N R N N NH N R A O H3C N O N N H O R G NH N O R C T The 3’-phosphorous group: phosphoramidite HO O B DMTr-Cl pyridine HO Base is protected DMTrO O B N P O CN DMTrO O B Cl HO pyridine O NC O P N[CH(CH3)2] 443 224 Automated, solid-phase DNA synthesis S I L I C A S I L I C A OH OH OH S I L I C A O Si (CH2)3 NH2 O O O O O Si (CH2)3 N C (CH2)2 C OH H O O DMT-O B2 O DMT-O S I L I C A B1 N O O HO O O I2, H2O CN O O oxidation of P N (iPr)2 CN DMT-O B2 B3 O O O P O coupling O S B1 P O NH N 5'- deprotection repeat cycle O O N O DMT-O DMT-O B1 Cl3CCOOH Si (CH2)3 N C (CH2)2 C O H O O O O P O O CN Cl3CCOOH 5'- deprotection B1 B O P O CN O B2 O O O S S O O O O P O CN O B1 O HO B3 HO B3 O Cl3CCOOH 5'- deprotection O S O O P O CN O NH3, H2O, ! B2 deprotect P and the base amino groups O O O P O CN O B1 O O P O O B2 O O O P O O B1 O S O O O 444 OH 28.17 Polymerase Chain Reaction (PCR): method for amplifying DNA using DNA polymerase and cycling temperature Heat stable DNA Polymerases (from archaea): Taq: thermophilic bacteria (hot springs)- no proof reading Pfu: geothermic vent bacteria- proof reading Mg 2+ two Primer DNA strands (synthetic, large excess) one sense primer and one antisense primer one Template DNA strand O O O -O P O P O P O O B dNTP’s OOOHO KARY B. MULLIS, 1993 Nobel Prize in Chemistry for his invention of the polymerase chain reaction (PCR) method. 445 225 A typical PCR temperature cycle Denaturation: 94 °C 0.5 - 1 min Annealing: 55-68 °C 0.5 - 1 min 5 °C below the Tm of the primer Extension: (replication) 72 °C 1 min + 1 min per Kb of DNA # of cycles 25 - 35 Final extension 72 °C 10 min 1 x 2 = 2 x 2 = 4 x 2 = 8 x 2 = 16 x 2 = 32 x 2 = 64 x 2 = 128 x 2 = 256 x 2 = 512 x 2 = 1,024 x 2 = 2,048 x 2 = 4,096 x 2 = 8,192 x 2 = 16,384 x 2 = 32,768 x 2 = 65,536 x 2 = 131,072 x 2 = 262,144 x 2 = 524,288 x 2 = 1,048,576 In principle, over one million copies per original, can be obtained after just twenty cycles 446 Polymerase Chain Reaction 5' 3' 3' 5' 5' 3' 3' 5' 95 °C denaturation 5' 3' 72 °C Taq, Mg 2+, dNTPs 55 - 68 °C 3' 3' anneal (+) and (-) primers extension 5' 3' 5' 3' 3' 5' 5' 3' denaturation 3' 5' 2nd cycle 5' 3' 3' 5' 5' 3' 3' 5' 95 °C 2 copies of DNA repeat temperature cycles amplification of DNA For a PCR animation go to: http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/pcr.html http://users.ugent.be/~avierstr/principles/pcrani.html 447 226