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Nucleic Acid Structure • • • • Dr. Asad Vaisi-Raygani Associate professor Department of Clinical Biochemistry, faculty of Medicine, Kermanshah University of Medical Sciences Nucleic Acids / Bases/Structure Imidazole By the attachment of different groups to the rings, different types of Py and Pu are generated. NA / Bases/ Classification • Purine Bases (pu): • Major : (A)&(G) • Minor: Inosine(I) & methyl guanine(7mG) • Unnatural : Mercaptopurine, Allopurinol & 8-Azaguanine NA / Bases/ Classification/ Py Hot spot • Pyrimidines (py): • Major : (T), (C) & (U) •Minor: DHU , 5mC & 5hmC •Unnatural: Fluorouracil (5FU) & 6-aza cytosine( AZC) Nucleosides N-base Nucleoside Nucleotide 9 9 1 1 1 Diversity RNA>>>DNA RNA have Enzyme activity • The oxo and amino groups of purines and pyrimidines exhibit keto-enol and amineimine tautomerism •but physiologic conditions strongly favor the amino N-glycosidic bond • Both therefore exist as syn or anti conformers (Figure 33–5). While both conformers occur in nature, anti conformers predominate. Energy carrier ( ATP & GTP) Regulatory roles: Group transfer • Methylated heterocyclic bases of plants include the • xanthine derivatives caffeine of coffee, • theophylline of tea, and theobromine of cocoa 7 1 Coenzymes (NAD, FAD & Co-A) SYNTHETIC NUCLEOTIDE ANALOGS ARE USED IN CHEMOTHERAPY • The purine analog allopurinol, used in treatment of hyperuricemia and gout, inhibits purine biosynthesis and xanthine oxidase activity. • Finally, azathioprine, which is catabolized to 6-mercaptopurine, is employed • during organ transplantation to suppress immunologic rejection. Nucleotides Absorb Ultraviolet Light • While spectra are pH-dependent, at pH 7.0 all the common nucleotides absorb light at a wavelength close to 260 nm. • The concentration of nucleotides and nucleic acids thus often is expressed in terms of “absorbance at 260 nm.” • 1 A260 unit of dsDNA=50 µg/ml H2o Chemical properties of nucleic acids: Nucleic acids show strong UV absorption at 260 nm due to the conjugated bases. (Detection method.) POLYNUCLEOTIDES 3’ OH 5’ phosphate • Phosphodiesterases rapidly catalyze the hydrolysis of phosphodiester bonds whose spontaneous hydrolysis is an extremely slow process. • Consequently, DNA persists for considerable periods and has been detected even in fossils. • RNAs are far less stable than DNA since the 2′-hydroxyl group of RNA (absent from DNA) functions as • a nucleophile during hydrolysis of the 3′,5′-phosphodiester bond. REDUCTION OF RIBONUCLEOSIDE DIPHOSPHATES FORMS DEOXYRIBONUCLEOSIDE DIPHOSPHATES • Reduction of the 2′-hydroxyl of purine and pyrimidine ribonucleotides, catalyzed by the ribonucleotide reductase complex (Figure 34–5), forms deoxyribonucleoside diphosphates (dNDPs). • The enzyme complex is active only when cells are actively synthesizing DNA. • Reduction requires thioredoxin, thioredoxin reductase, and NADPH. REDUCTION OF RIBONUCLEOSIDE DIPHOSPHATES FORMS EOXYRIBONUCLEOSIDE DIPHOSPHATES Nucleic Acid: 1-DNA 2-RNA DNA • led Watson, Crick, and Wilkins to propose in the early 1950s a model of a double- stranded DNA molecule • Deoxynucleotide • (A = T), • (G = C), • Because of the phosphate • moiety, they have acidic • character (negatively charged Rosaline, Wilkins (1950s) ‒ X-ray diffraction pattern of DNA. • The two strands, in which opposing bases are held together by hydrogen bonds, wind around a central axis in the form of a double helix. • Double-stranded DNA exists in at least six forms (A–E and Z). • The B form is usually found under physiologic conditions (low salt, high degree of hydration). Chemical properties of nucleic acids: Hydrophilic backbone (phosphate and pentose residues) and hydrophobic bases ⇒ base stacking (van der Waals and dipole-dipole interactions) • Right handed because • clockwise direction. • In the double-stranded molecule, restrictions imposed by the rotation about the hosphodiester bond, • the favored anti configuration of the glycosidic bond • and the predominant tautomers of the four bases (A, G, T, and C) • allow A to pair only with T • and G only with C,. 35.4 A Structural Stabilization? 1) Electrostatic interactions: negatively charged phosphate groups are located at the outer surface where they can be neutralized by: a) histones, b) cations (Mg2+), c) polyamines. 2) Base interactions: a) hydrogen bonds between bases (dipole-dipole interactions) b) stacking interaction between the planes of adjacent base pairs (van der Waals forces and dipole-dipole) Different bonds and interactions in • Covalent: PDE bonds in the backbone • Hydrogen: between complementary bases Primary or Natural (Watson-Crick) Secondary or Hoogsteen pairing (non-Watson-Crick) • Hyrophobic (van der Waals) between the stacked adjacent base pairs. DNA / General facts • Has specific groove (s) • It is flexible about its long axis It • It may be linear or circular DNA duplex can exist in different 3-D forms. B-form : the Watson-Click structure, standard form, most stable under physiological conditions A-form : a dehydrated form Z-form : left handed A short Z-DNA tracts may play a role in gene regulation. template (sense) strand or non coding template (sense) strand coding strand (non sense) or non template The Denaturation (Melting) of DNA Is Used to Analyze Its Structure • Concomitant with this denaturation of the DNA molecule is an increase in the optical absorbance of the purine and pyrimidine bases—a phenomenon referred • to as hyperchromicity of denaturation. • The midpoint is called the melting temperature, or Tm. • The Tm is influenced by the base composition of the DNA and by the salt concentration of the solution. • DNA rich in G–C pairs, which have three hydrogen bonds, melts at a higher temperature than that rich in A–T pairs, which have two hydrogen bonds. • THE CHEMICAL NATURE OF RNA DIFFERS FROM THAT OF DNA • Ribonucleic acid (RNA) is a polymer of purine and pyrimidine >90% NA <10% DNA • RNA can be hydrolyzed by alkali to 2′,3′ cyclic diesters of the mononucleotides, • compounds that cannot be formed from alkali-treated DNA because of the absence of a 2′-hydroxyl group. • The alkali liability of RNA is useful both diagnostically and analytically. Kind of RNA • mRNAs. • transfer RNAs; tRNAs • Many other cytoplasmic RNA molecules (ribosomal RNAs; rRNAs) • Some RNA molecules have intrinsic catalytic activity. • The activity of these ribozymes often involves the cleavage of a nucleic acid. • In all eukaryotic cells there are small nuclear RNA (snRNA) species that are not directly involved in protein synthesis but play key roles in RNA processing. • These relatively small molecules vary in size from 90 to about 300 nucleotides (Table 35–1). • small cytoplasmic RNA (scRNA) • Signal recognition particle • Concentration rRNA>tRNA> mRNA • diversity mRNA>tRNA>rRNA • The heterogeneous nuclear RNA (hnRNA) molecules may have a molecular weight in excess of 107, • whereas the molecular weight of mRNA molecules is generally less than 2 ×106. B. TRANSFER RNA (TRNA) • tRNA molecules vary in length from 74 to 95 nucleotides. • The tRNA molecules serve as adapters for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. • There are at least 20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein synthesis. 74-95 nts • The tRNA-appropriate amino acid is attached to the 3′-OH group of the A moiety of the acceptor arm. • The D, TC, and extra arms help define a specific tRNA. • template but must be formed by processing from a precursor • Although tRNAs are quite stable in prokaryotes, they are somewhat less stable in eukaryotes. • The opposite is true for mRNAs, which are quite unstable in prokaryotes but generally stable in eukaryotic organisms. SPECIFIC NUCLEASES DIGEST NUCLEIC ACIDS • Those which exhibit specificity for deoxyribonucleic acid are referred to as deoxyribonucleases. • Those which specifically hydrolyze ribonucleic acids are ribonucleases. • Within both of these classes are enzymes capable of cleaving internal phosphodiester bonds to • produce either 3′-hydroxyl and 5′phosphoryl terminals or • 5′-hydroxyl and 3′-phosphoryl terminals. • These are referred to as endonucleases. • There exist classes of endonucleases that recognize specific sequences in DNA; the majority of these are the • restriction endonucleases, • which have in recent years become important tools in molecular genetics and medical • Some nucleases are capable of hydrolyzing a nucleotide only when it is present at a terminal of a molecule; • these are referred to as exonucleases. • Exonucleases act in one direction (3′ → 5′ or 5′ → 3′) only. Histones Are the Most Abundant Chromatin Proteins • Nucleosomes or core histones • In the nucleosome, the DNA is supercoiled in a left handed helix over the surface of the disk-shaped histone octamer (Figure 36–2). Metaphase overall DNA Transcriptionally inert 10000 fold condence These four core histones are subject to at least five types of covalent modification: • DNA in active chromatin contains large regions (about 100,000 bases long) that are sensitive to digestion by a nuclease such as DNase I. • Hypersensitive sites(100-300bp) often provide the first sign about the presence and location of a transcription control element. SOME REGIONS OF CHROMATIN ARE “ACTIVE” & OTHERS ARE “INACTIVE Euchromatin [Transcriptionally active] Chromatin Structural genes, rRNA genes, regulatory regions, etc. Heterochromatin [Transcriptionally inactive] Heterochromatin [Transcriptionally inactive] •constitutive and facultative. constitutive Centromeric chromatin Attachment sites to nuclear matrix telomeres. • Facultative heterochromatin is at times condensed, • but at other times it is actively transcribed and, • thus, uncondensed and appears as euchromatin. • Chromatin containing active genes (ie, of the two members of the X chromosome pair in mammalian females, • one X chromosome is almost completely inactive transcriptionally and is heterochromatic. • However, the heterochromatic X chromosome decondenses during gametogenesis and becomes transcriptionally active during early embryogenesis—thus, facultative heterochromatin. • it is • The centromere is an adeninethymine (A–T) rich region ranging in size from 102 (brewers’ yeast) • to 106 (mammals) base pairs. It binds several proteins with high affinity. • This complex, called the kinetochore, • provides the anchor for the mitotic spindle. • It thus is an essential structure for chromosomal segregation during mitosis. • The function of the intervening sequences or introns, is not clear. 24% of the total human genome • They may serve to separate functional domains (exons) of coding information in a form • that permits genetic rearrangement by recombination to occur more rapidly than if • all coding regions for a given genetic function were contiguous. • humans have < 100,000 proteins • encoded by the ~1.1% of the human genome that is composed of exonic DNA. • This implies that most of the DNA is noncoding • The DNA in a eukaryotic genome can be divided into different “sequence classes.” • These are uniquesequence,or nonrepetitive, DNA and • These different genes represent unique DNA sequences that are present in single copies or at most only a few copies per genome • • repetitive sequence DNA. • Least 30% of the Genome • These are divided into two major classes: • middle repetitive (<10 copies per genome) • highly repetitive (>10 copies per genome) sequences • Some middle repetitive DNA consists of genes that specify • transfer RNAs, • ribosomal RNAs, • or histone proteins that are required in large amounts in the cell. • Other middle repetitive DNA sequences have no known useful function, • but may participate in DNA strand association and • chromosomal rearrangements during meiosis. • The highly repetitive sequences consist of 5–500 base pair lengths repeated many times in tandem. • These sequences are usually clustered in centromeres and telomeres of the chromosome and are present in about 1–10 million copies per haploid genome. • These sequences are transcriptionally inactive • and may play a structural role in moderately repetitive sequences, which are defined as • The • • • • being present in numbers of less than 106 copies per haploid genome, are not clustered but are interspersed with unique sequences. In many cases, these long interspersed repeats are transcribed by RNA polymerase II and contain caps indistinguishable from those on mRNA. Microsatellite Repeat Sequences • Satellite DNA consists of clusters of short, speciesspecific, • nearly identical sequences that are tandemly repeated hundreds of thousands of times. • AC repeats are very useful in constructing genetic linkage maps. • Most genes are associated with one or more microsatellite markers, • so the relative position of genes on chromosomes can be assessed, • as can the association of a gene with a disease. • Using PCR, a large number of family members can be rapidly screened for a certain microsatellite polymorphism. • These clusters are deficient in protein-coding genes and are found principally • near the centromeres of chromosomes, • suggesting that they may function to align the chromosomes during • cell division to • facilitate recombination • Trinucleotide sequences that increase in number (microsatellite instability) can cause disease. • The unstable p(CGG)n repeat sequence is associated with the • fragile X syndrome. • Other trinucleotide repeats that undergo dynamic mutation (usually an increase) are associated with • Huntington’s chorea (CAG), myotonic dystrophy (CTG), ONE PERCENT OF CELLULAR DNA IS IN MITOCHONDRIA • An important feature of human mitochondrial mtDNA is that because all mitochondria are • contributed by the ovum during zygote formation • it is transmitted by maternal nonmendelian inheritance. • Thus, in diseases resulting from mutations of mtDNA, • an affected mother would in theory pass the disease to all of her children • but only her daughters would transmit the trait. • However, in some cases, deletions in mtDNA occur during oogenesis and • thus are not inherited from the mother. • A number of diseases have now been shown to be due to mutations of mtDNA. • These include a variety of myopathies, • neurologic disorders, • some cases of diabetes mellitus .