Nucleic Acid Lecture Notes PDF

Nucleic acid Waraphan Toniti  Learning objectives Apply bioinformatics tools for structure and function of NA Explain physical and chemical properties of NA and its application Determine NA, AA, and protein relationship...

Nucleic acid Waraphan Toniti  Learning objectives Apply bioinformatics tools for structure and function of NA Explain physical and chemical properties of NA and its application Determine NA, AA, and protein relationship 2  Nucleic acid Nitrogenous base Pyrimidine Purine Pentose sugar Ribose deoxyribose Nucleoside Nucleotide 3  Nitrogenous base * * 4  Pyrimidine Thy Cyt Ura 5  Purine Ade Gua 6  Tautomerization Aromatic bases consequence for structures, electron distribution, and light absorption of nucleic acids. 7  Tautomerization Electron delocalization among atoms in the ring gives most of the bonds partial double-bond character.* *Lehninger 8 9  Pentose sugar 2’-deoxy-D-ribose D-Ribose 10  Conformation of ribose Puckered conformations: Endo-movement of C-atom closer to O-atom Exo-movement of C-atom away from O-atom C-2’ endo and C-2’ exo C-3’ endo and C-3’ exo 11  Nucleoside Nitrogenous base + pentose Adenosine (A) Deoxyadenosine (dA) Guanosine (G) Deoxyguanosine (dG) Deoxythymidine (dT)* Cytidine (C) Deoxycytidine (dC) Uridine (U)** *DNA 12 **RNA  Nucleoside (cont.) N--Glycosidic linkage 9 1 HO-H A dT 13  Nucleotide (cont.) Nitrogenous base + pentose + Pi Adenylate (adenosine 5’-monophosphate; AMP) Deoxyadenylate (2’-deoxyadenosine-5’-monophosphate; dAMP) Guanylate (guanosine 5’-monophosphate; GMP) Deoxyguanylate (2’-deoxyguanosine-5’-monophosphate; dGMP) Deoxythymidylate (2’-deoxythymidine-5’-monophosphate; dTMP)* Cytidylate (cytidine 5’-monophosphate; CMP) Deoxycytidylate (2’-deoxycytidine-5’-monophosphate; dCMP) Uridylate (uridine 5’-monophosphate; UMP)** *DNA 14 **RNA Absorption spectra of the common nucleotides. 15  Nucleotide (cont.) Ester bond Acid-anhydride bond 16  Dinucleotide, oligonucleotide, polynucleotide Phosphodiester linkage between 5’-phosphate group of one nucleotide unit and 3’-hydroxyl group of the next nucleotide Nicotinamide adenine dinucleotide (NAD+) Nicotinamide adenine dinucleotide phosphate (NADP+) Flavin mononucleotide (FMN) or riboflavin-5’-phosphate Flavin adenine dinucleotide (FAD+) 17  Dinucleotide, oligonucleotide, polynucleotide (cont.) Phosphodiester bond 18 NAD+ vs NADH 19 CPILE-a:NAD+ (5WTZ) CPILE-a:NADH (5WU0) 20  5WTZ 5WU0 PDB ID: 5WTZ, 5WU0 21 22 FAD vs FADH2 23  Nucleic acid Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) DNA  protein Base composition varies among species Same species, same base composition Age, nutritional stage, or environment not change base composition 24  Nucleic acid (cont.) Complementary bases; A-T or A-U G-C Chargaff’s rules A=T C=G Then A + G = T + C 25 Nucleic acid (cont.) 26  DNA RNA Double-stranded Single-stranded 2’-deoxyribose Ribose A, T, G, C A, U, G, C Rapidly hydrolysis under alkaline condition mRNA, rRNA, tRNA 27  X-ray crystallography https://kaiserscience.files.wordpress.com/2018/01/dna-x-ray-crystallography.png 28  Watson-Crick model DNA consists of two antiparallel helical strands rotate around the same axis. The hydrophilic backbones are on the outside of the helices while the hydrophobic purine and pyrimidine bases are stacked inside. The deoxyribose is in the C-2’ endo conformation. The offset pairing of the two strands creates a major groove and minor groove on the surface of the duplex. 29  DNA backbone 5’ end Hydrophilic backbone Hydroxyl group of deoxyribose Ionized phosphate group Negatively charged at pH 7 Hydrophobic bases Hydrophobic stacking interaction Van der Waals interaction + dipole-dipole interaction 3’ end 30 31 32  At least three forms of DNA can be crystallized. B-form* anti right-handed double helix C-2’ endo 10.5 bp/ turn major groove and minor groove *Watson-Crick model is referred to B-form DNA. 33  B_A_Z conformation A-form anti right-handed double helix C-3’ endo 11 bp/ turn 34  B_A_Z conformation (cont.) Z-form anti for pyrimidine and syn** for purine left-handed double helix C-2’ endo for pyrimidines, C-3’ endo for purines zigzag appearance of the backbone 12 bp/ turn **C-2’ endo for pyrimidines, C-3’ endo for purines 35 36  Palindromes and mirror repeats Palindromes: twofold symmetry Mirror repeats: symmetry within each strand 37 38  Nucleic acid properties Native DNA are highly viscous at pH 7.0, 25 oC. How about extremely changes in pH and temperature? What is the consequence? 39  Denaturation and renaturation of DNA High temperature caused melting of DNA. Hydrogen bonds disruption Two single strands Melting point; tm The higher G-C base pairs, the higher melting point of the DNA Annealing occurs when the temperature or pH return to the normal range. Double strand DNA hybridization 40  Genetic information DNA serves to store genetic information. Template strand; codogenic strand (-) Sense strand; coding strand (+) Genes carry out structural and/or catalytic tasks. Codons: A, G, C, T Triplet codons: amino acid RNAs involve in protein synthesis. Codons: A, G, C, U 41  RNA Single strand Right-handed helix Base-stacking interaction 42  Secondary structure of RNA Hairpin loop Double-stranded Complex loops 1GID 43  Nucleic acid (cont.) RNA Messenger RNA (mRNA) Transcription Transfer genetic information from DNA to ribosome Ribosomal RNA (rRNA): RNA + protein Small subunit Large subunit Transfer RNA (tRNA) Translation Transfer genetic information from mRNA to protein 44  Ribosome Three binding sites: A, P, and E site. A site binds to an aminoacyl-tRNA At the P site, the aminoacyl-tRNA + peptidyl-tRNA forming a new peptide bond. The tRNA carry on the last amino acid is moved to the E site 45 46  rRNA Type Size Large subunit Small subunit Prokaryote 70S 50S (5S, 23S) 30S (16S) Eukaryote 80S 60S (5S, 28S) 40S (18S) 47 Structure of tRNA 48  A plasmid editor (ApE) Read and modify DNA sequences Mutation Deletion Addition Read ABI sequencing files 49 The central dogma of molecular biology Replication DNA Transcription Reverse transcription RNA Replication Translation Protein 50  DNA replication DNA template One strand is the complement of the other. Each strand provides the template for a new, complementary sequence. Semiconservative 51  DNA replication (cont.) Replication forks Dynamic points DNA unwind Origin Unique point Starting point 52  DNA replication (cont.) Simultaneously, bidirectional event A new strand of DNA is always synthesized in the 5’ to 3’ direction. Leading strand Continuous strand Lagging strand Discontinuous strand Okazaki fragments 53  DNA replication (cont.) DNA helicases To unwind double- or single-stranded DNA and/or RNA chains By hydrogen-bond breaking Topoisomerase To relief topological stress DNA-binding protein 54  DNA polymerases DNA polymerases To polymerize DNA By transferring phosphoryl group (dNMP)n + dNTP (dNMP)n+1 + Ppi 55 56  DNA polymerases (cont.) Template At least single strand! Primer A strand segment which complements to the template. Forward primer Reverse primer Reverse complement primer 57  DNA primer Forward primer = ATG Reverse primer = ATT Reverse complement primer = ? https://www.bioinformatics.org/sms/rev_comp.html 58  Proofreading Nucleases (DNases) Exonucleases At the end of DNA 5’ to 3’ OR 3’ to 5’ Endonucleases* At specific internal sites *restriction enzymes 59 https://www.snapgene.com/resources/plasmid-files/?set=pgex_vectors_(ge_healthcare)&plasmid=pGEX-4T-1 60  Proofreading (cont.) Active site geometry fit to A-T and G-C standard base pairs. Klenow fragment of DNA polymerase I from E. coli. First reported in 1970, it retains the 5' → 3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity*. *remove 5’ to 3’ exonuclease domain 61 Klenow fragment Zn 1KLN 62  5’ 3’ 5’ 3’ 63  DNA repair systems Mismatch repair Strand discrimination by template methylation All adenines within 5’GATC are methylated. Base-excision repair Cleave the N-glycosyl bond AP site (abasic site) AP endonuclease Nucleotide-excision repair Exinuclease hydrolyzes two phosphodiester bond Direct repair 64  DNA supercoil Chromosomal elements Genes = information for functional polypeptides, RNAs Introns = noncoding segments Exons = coding segments DNA supercoiling The coiling of a coil Topological property 65 Coil and Supercoil 66  Genes and Chromosomes Genes are segments of DNA that code for polypeptide chains and RNAs. A portion of chromosome that determines specific character = gene One gene-one polypeptide (/RNA*) Genotype  Phenotype 67  Viruses DNA virus RNA virus B-form double helix Mixture of single strand and double strand Accurate replication Error-prone replication Protected by cell Actively degraded by cell (dsRNA) Packaged into preformed capsid Co-assembles with capsid protein shell 68  Example of Viruses (cont.) DNA virus RNA virus dsDNA Poxviridae ssRNA (-) Orthomyxoviridae Herpesviridae Paramyxoviridae Polyomaviridae Rhabdoviridae Papillomaviridae Adenoviridae ssDNA Circoviridae ssRNA (+) Caliciviridae Parvoviridae Picornaviridae Flaviviridae Coronaviridae Togaviridae 69  Penton Hexon Minor capsid proteins (A) Cartoon view of adenovirus, highlighting the major capsid proteins as labelled. (B) Structural view of an adenovirus vertex modelled from CryoEM structure (PDB ID: 6B1T). 70 https://doi.org/10.3390/cancers10060201  Prokaryotes Bacteria Chromosome Single double-stranded circular DNA Extrachromosomal elements plasmids 71 Prokaryote (cont.) Chromosome Plasmids } 72  Eukaryotes Number of chromosomes Escherichia coli 1 Drosophila melanogaster (fruit fly) 18 Oryza sativa (rice) 24 Homo sapiens sapiens 46 Canis lupus familiaris 78 Columba livia (pigeon) 80 73  Eukaryotes (cont.) Nuclear chromosome Diploid (2n) Mitochondrial DNA (mtDNA) Circular duplex Chloroplast DNA (cpDNA) Primary endosymbiosis Cyanobacteria 74 DNA and Chromosomes 75 Chromatin 76  DNA and Chromosomes (cont.) p arm Centromere q arm 77  DNA and Chromosomes (cont.) Telomeres (cap) at the end of chromosome by x(TTAGGG) and form T-loop During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, so in each duplication the end of the chromosome is shortened. Telomerase, which carries out the task of adding repetitive nucleotide sequences to the ends of the DNA. Telomerase, thus, "replenishes" the telomere "cap" of the DNA. 78 Telomere shorten until senescence! 79 Domestic dog karyotypes 80  Mouse karyotypes Cat karyotypes 81  Cell cycle G0 phase S phase Resting cell/ quiescent DNA synthesis Withdraw from cell cycle RNA and protein synthesis G1 phase G2 phase RNA and protein synthesis RNA and protein synthesis M phase Mitosis and cytokinesis 82  Cell cycle G0 83  Cell division Interphase G1, S, and G2 phase Mitosis Prophase Metaphase Anaphase Telophase 84  References Lehninger Principles of Biochemistry Proceedings of the National Academy of Sciences, Vol. 65, No. 2, pp. 168- 175, January 1970. THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 274, No. 25, Issue of June 18, pp. 17395–17398, 1999. https://doi.org/10.3390/cancers10060201 PDB DOI: https://doi.org/10.2210/pdb1dpi/pdb PDB DOI: https://doi.org/10.2210/pdb1KLN/pdb 85  References https://jorgensen.biology.utah.edu/wayned/ape/ 86