Polysaccharides ( glycans ) are biopolymers that consist of several hundreds up to many thousands of monosaccharide units linked into chains through or O -glycosidic bonds. The monosaccharide units may be linked linearly, or the chains may be branched. Polysaccharides are polydisperse polymers, they consist of chains different in length. Due to the high molecular mass, polysaccharides either don‘t dissolve in water or form viscous colloid dispersions. Polysaccharides are nonreducing compounds – in the molecules of which containing a high number of monosaccharide units, there is (if any) only a single reducing group. Classification of polysaccharides Homopolysaccharides (homoglycans) - give a single monosaccharide on complete hydrolysis Heteropolysaccharides (heteroglycans) - give two or more different monosaccharides, if hydrolyzed completely (e.g. starch, glycogen, cellulose, chitin, inulin, pectins) (e.g. acidic glycosaminoglycans, plant gums, and mucilages, agar)
Homopolysaccharides (homoglycans) is the energy-storing polysaccharide of plants , the form in which glucose is stored by plants for later use. Partial hydrolysis of starch gives maltose , and complete hydrolysis gives only glucose . In water, starch granules swell and form viscous colloid dispersion by heating. Starch can be separated by various techniques into two fractions: Amylose constitutes about 20 - 30 % of common starch types and forms unbranched helical continuous chains comprising 200 -1000 D -glucopyranose units, with α(1 4) glycosidic linkages. Amylopectin is the main and highly branched component of starch (70 – 80 %). Each molecule may contain 300-6000 D -glucopyranose units. Chains with consecutive α(1 4) glycosidic bonds are connected at branch points by α(1 6) linkages. Starch ( Lat. amylum )
Amylose ( 1 ->4 )- - D -glucan (glucopyranan) the nonreducing end the free anomeric hydroxyl (the reducing end)
Amylopectin Chains with consecutive (1 ->4) links average only about 30 units in length. B ranch points occur every 8 – 12 glucose residues. -1,6-glycosidic linkage at the branch point 6 1 -1,4-glycosidic linkages 4
is the energy-storing polysaccharide in animals . Like amylopectin of starch, it is made of α(1 4) and α(1 6) linked glucose units. Glycogen has a higher molecular mass than amylopectin, starch (up to 100 000 glucose units), and its structure is even more branched. Glycogen Glycogen is present in the cytosol of animal cells in the form of granules ranging in diameter from 10 to 40 nm. The two major sites of glycogen storage are the liver (about 5 %) and skeletal muscle (about 1 %). The core of the glycogen particle is a protein (glycogenin, G).
Cellulose ( 1 -> 4 )- - D - glucan (glucopyranan) The unbranched chains of macromolecules (up to 5 000 glucose units) aggregate to give fibrils , in which chains are bound by intra- and intermolecular hydrogen bridges. These fibrils wound spirally in opposite directions around the central axis to form cellulose fibres .
Cellulose is not soluble in water. It serves as a extracellular structural element in plants . Cotton, wood, hemp, linen, straw, and corncobs are mainly cellulose. Humans and other animals cannot digest cellulose , their digestive systems lack the enzymes that can catalyze the hydrolysis of β-glycosidic bonds. Many bacteria , however, do contain β-glucosidases and can hydrolyze cellulose. Ruminants (cud-chewing animals) have such bacteria in their rumen, as well as, for example, termites, so that those animals thrive on cellulose as their main food. Cellulose is the major component of insoluble dietary fibre ( roughage ). Fruit, vegetables 1 – 2 % Cereals, pulses 2 – 4 % White flour 0.2 – 3 % Cereal husks (bran) 30 – 35 % Cellulose in some foodstuffs
Dietary fibre (roughage) is not a nutrient, though it is important to take in sufficient amounts of it. Roughage occurs solely in food of plant origine. It is a mixture of cellulose and other plant biopolymers (both polysaccharides and lignin) that cannot be hydrolyzed by the digestive enzymes in the small intestine . According to the solubility of dietary fibre components in water, two types are distinguished: - indissolvable part – cellulose, some hemicelluloses and lignin, which may be found in feces without being decomposed, and - soluble components – pectins, plant gums and slimes, soluble hemicelluloses; majority of them is fermented by the enzymes of microflora during the passage through the large intestine . Dietary fibre - supports peristalsis of the intestine by increasing the volume of stools, - helps to eliminate cholesterol from the body by preventing the reabsorption of bile acids, - slows down intestinal absorption of ingested saccharides as well as other nutrients, etc.
Inulin Inulin is the energy-storing unbranched polysaccharide (up to 100 D -fructose units) , which occurs in tubers of some plants (topinambour, sunflower, chicory) and also in small amounts in fruit and vegetables. It is one of the components of dietary fibre. ( 2 ->1 )- β - D - fructan (fructofuranan) nonreducing end β ( 2->1 )
Dextran branching (1 3) (1 6) bonds is a polysaccharide (unusual D -glucan) produced by some species of bacteria from sucrose. The molecular mass of native dextrans is in the range 10 4 – 10 6 . Viscous colloid solutions of dextran can be used as short term substitutes for blood plasma (acute bleeding, burns). By chemical cross-linking, dextrans are modified to gels that serve as " molecular sieves“ in laboratories (gel filtration, gel chromatography). Dextran formed from saccharose by the action of oral microflora is also a insoluble component of dental plaques that cannot be decomposed by the -amylase of saliva.
Heteropolysaccharides (heteroglycans) give two or more different monosaccharide units on complete hydrolysis (monosaccharides, amino sugars and/or uronic acids). Those units are linked by glycosidic bonds. The most important group of heteropolysaccharides are glycosaminoglycans . Numerous heteropolysaccharides are known, such as plant gums and mucilages
3) Glc NAc (β1–4) Glc A (β1–3) Glc NAc (β1–4) Glc A (β1 Segment of the hyaluronic acid molecule The length of hyaluronate linear unbranched chain can reach 4000 nm. [ N -Ac-glycosamine glycuronic acid ] n Glycosaminoglycans The common feature of various glycosaminoglycans (e.g., hyaluronic acid, heparin, dermatan sulfate) is the regular alternating of disaccharidic units - N -acetylated amino sugars and glycuronic acids. Alcoholic groups of both types of monosaccharidic units are oft sulfated (except hyaluronic acid).
Common constituents of glycosaminoglycans : α - D - glucuronic acid - D- galacturonic acid N- sulfated glucosamine - L - iduronic acid (5-epimer of glucuronic acid) N - Ac-galactosamine 4-sulfate N - Ac-glucosamine 6-sulfate
Hyaluronic acid Heparin Chondroitin 4-sulfate 6-sulfate Dermatan sulfate Keratan sulfate GlcNAc GlcA GlcNAc GlcA or IdoA sulfate GalNAc-4-sulfate GlcA -6-sulfate GlcA GalNAc GlcA or IdoA sulfate GalNAc sulfate Gal Major types of glycosaminoglycans ( GAG )
Heparin prevents blood clotting in vivo (antithrombotic activity) as well as in vitro (an anticoagulant in laboratories, uncoagulable blood or blood plasma) because of its ability to inactivate antithrombin. It is released from basophilic granules of mast cells. For therapeutic use, heparin is isolated from animal tissues. Heparin (1–4) L -Ido A -2-sulfate (1–4) Glc N -N,6-bissulfate ( 1–4)
Proteoglycans If not thinking of dense collagen connective tissue and bone, proteoglycans represent the most voluminous component of amorphous ground substance in connective tissue, which fill in the space among fibres and cells. In proteoglycans, numerous (very approximately 100) chains of different glycosaminoglycans (that include 10 –100 monosaccharide units) bind through glycosidic bonds the core protein forming so aggregates called monomeric proteoglycans or agrecans . Hyaluronic acid is the only glycosaminoglycan that occurs in the ground substance of connective tissue as a free heteropolysaccharide without any covalent bond to proteins. Other GAGs are attached to proteins through glycosidic bonds forming proteoglycans.
A large number of simple monomeric proteoglycans (agrecans) bind their globular domains of core proteins non-covalently to a long chain of hyaluronic acid . Huge aggregates are formed in this way namely in hyaline cartilages. They contribute to the resistance of a cartilage to mechanical pressure and to its elasticity . Proteoglycans are highly hydrated , and numerous carboxylate and sulfate groups bind due to negative electric charges large amounts of hydrated cations. Monomeric proteoglycan (agrecan) agrecans In spite of its large size , c ore protein of proteoglycans represents only about 5 – 15 % mass of the proteoglycan. The agrecan structure resembles a bottle-brush. hyaluronate
Oligosaccharides and polysaccharides (homopolysaccharides as well as heteropolysaccharides) are taken as homoglycosides . In homopolysaccharides, only saccharide units are bound mutually through glycosidic bonds. Heteroglycosides are glycosides in which a saccharide is linked with an non-saccharide component by means of glycoside bond. The non-saccharide components are called aglycones or genins . Saccharides are able to fo form a O -glycosidic link with alcohols, phenols, and acids (e.g. numerous natural products, some glycoproteins , glycolipids , products of detoxification of phenols, bilirubin, aromatic acids), N -glycosidic link with amines, amides, nitrogenous heterocycles (e.g. nucleosides ,some glycoproteins ), or S -glycosidic link with thiols (e.g. mustard oil glycosides in horse-radish root, radish, rape-seed, kohl-rabi with a pungent taste ).
Examples of naturally occuring (hetero)glycosides: Salicin glycosylated salicylalcohol Amygdalin benzaldehyde, HCN, disaccharide glycosyl Digitoxigenin Medicinals – digitoxin - purple foxglove plant – arbutin - cranberry leaves Poisons – amygdalin - fruit kernels of bitter almonds, apricots Flavours – vanillin - D -glucoside – sinigrin - horseradish roots Tensides – saponins (steroid aglycon) of soap roots, quillaja bark Dyes – indican (indigogen) - indigo plant – anthocyans - blue and red flowers etc.
Purine bases of nucleotides N N N N H NH 2 adenine 6-aminopurine, Ade N N N N H OH H 2 N guanine 2-amino-6-hydroxypurine, Gua hypoxanthine 6-hydroxypurine N N N N H OH xanthine 2,6-dihydroxypurine N N N N H HO OH N N N N H 1 2 3 4 6 7 8 9 5 purine atypical numbering!
uracil 2,4-dihydroxypyrimidine, Ura N N HO OH thymine 5-methyluracil, Thy N N CH 3 HO OH N N NH 2 HO cytosine 4-amino-2-hydroxypyrimidine, Cyt lactim form lactam form of uracil N N N N O O H H HO OH They exist in two tautomeric forms. Only the lactam forms can be ribosylated to give nucleosides: pyrimidine 1,3-diazine N N Pyrimidine bases of nucleotides
5-fluorouracil 6-mercaptopurine Synthetic analogs of bases Some analogs of bases inhibit the synthesis of purines and pyrimidines and so the biosynthesis of DNA; they are clinically useful anticancer drugs. Examples:
Nucleosides Nucleosides are heteroglycosides, glycosylated nitrogenous bases. Ribosylated and deoxyribosylated purine and pyrimidine bases are components of nucleotides, substrates of the biosynthesis of nucleic acids. In most nucleosides, D -ribose or 2-deoxy- D -ribose is attached through β- N -glycosidic bond to N 9 of purine bases or to N 1 of pyrimidine bases. ( ) Systematic names of nucleosides are the, e.g., 9-β- D -ribofuranosylguanine and N 1 -2'-deoxy-β- D -ribofuranosylthymine, but usually trivial names are preferred. - N -glycosidic bond N-base
Trivial names of nucleosides are derived from the names of bases by replacing their endings with the suffix –osine in purine nucleosides, and with the suffix –idine in pyrimidine nucleosides. Ribonucleosides Abbreviation Nucleoside symbol Adenosine Ado A Guanosine Guo G Inosine (base hypoxanthine) Ino I Xanthosine Xao X Cytidine Ctd C Uridine Urd U Ribothymidine Thd T Deoxyribonucleosides Deoxyadenosine dAdo dA Deoxyguanosine dGuo dG Deoxycytidine dCtd dC Thymidine dThd dT
Purine ribonucleosides xanthosine guanosine 9- β- D -ribofuranosylguanine inosine 9-β- D -ribofuranosyl hypoxanthine adenosine
Pyrimidine nucleosides deoxycytidine N 1 -2 '- deoxy -β- D -ribofuranosylcytosine uridine N 1 -β- D -ribofuranosyluracil cytidine N 1 - β- D -ribofuranosylcytosine thymidine N 1 -2 ' - deoxy -β- D -ribofuranosylthymine 5 pseudouridine ribothymidine
Synthetic nucleoside analogs N =N= N zidovudin (azidothymidine) aciclovir cytarabin ( N 1 - β- D -arabinosyl cytosine) Examples: Cytarabin is an effective cytostatic in hematooncology. Zidovudin is used in the treatment of HIV infection, because it inhibits the viral reverse transcriptase. Aciclovir inhibits DNA polymerase of herpes virus. Similarly to analogs of bases, some nucleosides with modified glycosyls are also useful in clinical medicine as inhibitors of the biosynthesis of nucleic acids.
Nucleotides are phosphate esters of nucleosides . Phosphate may be attached through ester bond at carbon atoms 5 ' , 3 ', or (in ribose only) 2'; the primed numbers indicate the position on glycosyl. The primary alcoholic group at carbon 5' may also bind diphosphate or triphosphate. Most nucleotides are nucleoside 5'-phosphates : 5 ' 3 ' ester bond 2 ' Nucleotides are water-soluble and acidic (all the bound phosphates are ionized). Di- and triphosphates have high affinity for Mg 2+ and Ca 2+.
Nucleotides are ubiquitous substances in living systems. Functions of nucleotides: – they are the monomeric units of nucleic acids, – nucleoside triphosphates are the energy-rich end products of energy-releasing pathways – ATP and other triphosphates serve oft in activating of metabolic substrates (phosphorylation, UDP glucose, acyl adenylates, etc), – have an important role in regulation of metabolism (intracellular concentrations of ATP and ADP, concentration of cAMP – a second messenger), – adenine nucleotides are components of various coenzymes (NAD, NADP, FAD, FMN, coenzyme A), etc.
Nomenclature of nucleotides Nucleotides are usually named as the phosphates of their nucleosides. Nucleoside 5 '-phosphates In their names, the numerical locant 5' may be omitted . E.g. , the simplest adenine nucleotide is adenosine monophosphate (AMP), then adenosine diphosphate (ADP) or triphosphate (ATP). For adenosine 5 ' -monophosphates, the names of acids are used sometimes. E.g., adenosine 5 ' -phosphate AMP adenylic acid (adenylate) uridine 5 ' -phosphate UMP uridylic acid (uridylate) deoxyguanosine 5 ' -phosphate dGMP deoxyguanylic acid (deoxyguanylate) deoxycytidine 5 ' -phosphate dCMP deoxycytidylic acid (deoxycytidylate) Nucleoside 3 ' - or 2 ' -phosphates – the locant for phosphate must be given. adenosine 3 ' -phosphate th ymidine 3 ' -phosphate
Adenosine 5 '- monophosphate ( AMP, adenylate ) Adenosine 5'-triphosphate ( ATP ) + + H + + H 2 O ADP H
Cyclic GMP ( cGMP ) has a similar specific function in transduction of extracellular signals across cytoplasmatic membranes, e.g. in photoreceptor cells (mechanism of vision). Cyclic adenosine 3',5'-monophosphate ( cAMP ) is the first known second messenger. It is formed from ATP in the cells, when some hormones or neurotransmitters bind onto their specific receptors on the surface of cytoplasmatic membranes. cAMP then activates phosphorylation of intracellular proteins, which evokes the cellular r espons. ATP diphoshate + AMP + H 2 O
NAD ( nicotinamide adenine dinucleotide ) and NADP ( NAD 2 ' -phosphate ) are coenzymes of dehydrogenases : adenine anhydride bond nicotinamide 2 ' NAD + (oxidized form)
FAD ( flavin adenine dinucleotide ) and FMN ( flavin mononucleotide ) are prosthetic groups of flavin dehydrogenases . Similarly to pyridine coenzymes NAD and NADP, they transfer two atoms of hydrogen: 6.7-dimethylisoalloxazine ribitol ( D -ribityl-) anhydride bond adenine coenzyme FAD (oxidized form)
Coenzyme A is a transporter of acyls (acetyl, fatty acyls, acyls that are products of 2-oxoacids decarboxylation, etc.). Acyls are transported in the form of thioesters: H O C H 2 C HS C H 2 C H 2 H N O C C H 2 C H 2 H N O C C H C H 3 C H 3 cysteamine pantothenic acid 3 ' - phosphoADP
Nucleic acids Deoxyribonucleic acids ( DNA ) and ribonucleic acids ( RNA ) are two classes of nucleic acids. Their functions are quite different. DNA is the hereditary molecule in all cellular life forms as well as in many (not all) viruses. The section of DNA molecule that encodes (contains information for the synthesis of) a unique functional protein is a structural gene. DNA has but two functions – to direct its own replication during cell division, and – to direct the transcription of complementary molecules of RNA . are polynucleotides – huge linear polymers consisting of nucleotides joined by 3 '-> 5 ' phosphodiester bonds .
RNA has more biological functions than DNA : – messenger RNA (mRNA) – the RNA transcripts of DNA sequences direct the ribosomal synthesis of proteins, – ribosomal RNA (rRNA) has a structural role (66 % of ribosomal mass are RNA molecules), – transfer RNA (tRNA) delivers amino acids to the ribosomes, – certain RNAs are associated with specific proteins (ribonucleo- proteins) and participate in posttranscriptional processing of other RNAs; (– in many viruses RNA (not DNA) carries the hereditary information.)
3 '-> 5 ' phosphodiester link between 3 ' -OH and 5 ' -OH Polynucleotide chain structure 5 ' -phosphate end free 3 ' -OH end (event. 3 ' -phosphate end) Bases are attached to the sugar- phosphate backbone through β- N -glycosidic bond s . By convention, direction of reading is from 5'-end to 3'-end.
P P 5 ' - P P P P 3 ' - OH Ura Cyt Ade Ade Gua Cyt RNA chain Symbolic notation of the base sequence From the 5 ' -end: p U -> C -> A -> A -> G -> C p U p C p A p A p G p C p U - C - A - A - G - C Identical chain from the 3 ' -end: 5 ' - UCAAGC C ←G←A←A←C←U p UCAAGC 3 ' - CGAACU Primary structure - abbreviated notation DNA chain p dG - dC - dT - dT - dG - dA d (p GCTTGA ) or d ( A ←G←T←T←C←G ) GCTTGA 3 ' - AGTTCG
Deoxyribonucleic acid One dsDNA molecule (one human haploid chromosome, chromatide), consists of (1 – 3) 10 8 base pairs (bp), the average value is 1.3 10 8 bp. The average relative molecular mass M r of two nucleotides equals 660, so that the molecular mass M r of a dsDNA molecule can reach 10 11 . DNA is surprisingly irregular in a sequence-specific manner. Nuclear DNA of eukaryotes is linear double-stranded DNA (dsDNA). The size of its molecules is generally enormous. Most sequences of nucleotides (about 70 %) are quite unique, but only 3 % code for proteins. The other are either moderately and highly repetitive (20 %) or in the form of inverted repeats (10 %, called satellite sequences). Mitochondrial DNA (mtDNA) is double-stranded and circular . Bacterial DNA is linear or circular dsDNA in the form of chromosome or plasmids. Some viruses contain single stranded DNA. Human mtDNA consists of only 16 500 base pairs, almost entirely without non-coding regions.
2 April 1953 J. D. Watson , F. H. C. Crick : " Molecular structure of nucleic acids. A Structure for Deoxyribose Nucleic Acid. " Nature (1953), Vol. 171, page 737 Secondary double helical structure of DNA 1962 Nobel Prize for Medicine to J.D. Watson, F.H.C. Crick, M.H.F. Wilkins “ ...for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material…”
<ul><li>Two polynucleotide chains wind about a common axis with a right-handed twist . Two strands are antiparallel , they run in opposite direction. </li></ul><ul><li>The coiling is plectonemic – the strands cannot be separated without unwinding the helix. </li></ul><ul><li>The hydrophilic ribose-phosphate chains are coiled about its periphery ( " sugar-phosphate backbone "); negatively charged phosphate groups bind positively charged groups of proteins and simple cations. </li></ul>Secondary double helical structure of DNA 5´-end 3´-end 5´-end 3´-end the main helical axis
The bases fill the inner of the helix as complementary base pairs – each base forms hydrogen bridges to the complementary base on the opposite strand. Hydrogen bonds originate spontaneously, without enzymatic catalysis. The planes of bases are nearly perpendicular to the helix axis as well as parallel. Top view: sugar-phosphate backbone
Pairing of bases (Watson-Crick geometry ) is the principle of chain complementarity: Non-Watson-Crick base pairs have relatively little stability, they don't occur in dsDNA. Such pairs, e.g. A–T pairs (Hoogsteen geometry) occur in the tertiary structure of tRNAs. In dsDNA, the number of Ade equals the number of Thy and the number of Gua equals the number of Cyt. On the other hand, the ratio Ade/Gua (equal to the ratio Thy/Cyt) characterizes individual dsDNAs. dRib dRib dRib dRib d G d C ( G ≡ C in RNAs) d A =d T ( A = U in RNAs)
Non-covalent interactions in dsDNA Attractions: – hydrogen bonds between bases – - interactions between adjacent base pairs (the cause of base "stacking", very important for the helix coherence) – hydrophobic interactions Repulsive forces: – repulsions between negatively charged phosphate groups dsDNA is a conformationally variable molecule , not the rigid helical column . Double helical conformations of DNA can be classified into three general families called the A -, B -, and Z - forms . Besides the typical forms, local deviations may occur with some bends, less or more tightly wound segments, short single-stranded loops, and even as cruciform structure (if palindromes are present). Conformation of dsDNA is irregular in a sequence-specific manner. Nevertheless, the repertoire of DNA secondary structures is limited, when compared with RNAs.
34 nm 3.4 nm 5´-end 3´-end 3´-end 5´-end 20 nm is the predominant form of dsDNA - the regular right-handed helix of Watson and Crick. The "ideal form": B-form of DNA (B-DNA) 10 base pairs per one turn Two unequal grooves arise on the surface because the glycosidic bonds of a base pair are not diametrically opposite each other.
Grooves are the sites of specific binding of regulatory molecules Phosphate groups on the grooves edges can bind positively charged aminoacyl residues of proteins. Each groove is lined by potential hydrogen-bond donor and acceptor atoms (N and O atoms of the bases) that enable sequence- specific binding of regulatory and other proteins. The major groove displays more features (on the top edges of base pairs) that distinguish one base pair from another than does the minor groove. Moreover, the larger size of the major groove makes it more accessible for interactions with proteins that recognize specific DNA sequences. major groove minor groove
A-DNA is a wider and flatter right-handed helix than B-form (s. No. 32). It has 11 base-pairs per one turn . Base pairs are inclined to the helix axis, the plane of base pairs may be tilted by as much as 20 °. The sugar rings are puckered differently (3´-endo conformation) from the way they are in the B-form (2´-endo). The major groove is narrow and deep, overlapped in part by phosphates. A-form of the double helix exists in the DNA/RNA hybrids and in double-helical RNA structures (arms of single-stranded RNAs). A-form of DNA (A-DNA) In vitro, B-form may be transformed reversibly into A-form by partial dehydration (relative humidity 75 %). In vivo , A-form of dsDNA may occur only in certain segments of helices.
Z-DNA is a left-handed double helix that has 12 Watson-Crick base pairs per turn. The line joining successive phosphate groups follows a zig-zag path around the helix (hence the name Z-DNA); the major groove is broad and flat and the minor groove very narrow so that it can be hardly discerned. Z-form occurs always in segments with alternating purine-pyrimidine base sequences. High ionic strength stabilizes Z-form, the methylation of cytosine residues promotes Z-DNA formation and initiates the separation of DNA strands. B- Z-
Comparison of DNA conformational forms B-form A-form Z-form
C(2´)-endo for Py C(3´)-endo for Pu C(3´)-endo C ( 2´ ) -endo Sugar pucker narrow and deep broad and shallow narrow and deep Minor groove flat narrow and deep wide and deep Major groove 7 20° 1 º - 6 º Base tilt from normal to the helix axis 45 nm 28 nm 34 nm Helical pitch (rise per turn) 3 0° 33° 36 º Helical twist per bp 12 11 10 Base pairs per helical turn 18 nm 26 nm 20 nm Diameter left-handed right handed right-handed Helical sense dsDNA B form A form Z form
Human nuclear genome consists of c ir ca 3 10 9 base pairs. 70 % of this number are unique sequences , which occur mostly in one copy in the haploid genome. Among those unique sequences, approx. 25 000 structural genes coding for proteins are included, as well as genes coding for structures of rRNA and tRNA . DNA sequences that code for proteins represent only 3% of genome. Moderately repetitive sequences (less than 10 6 copies) and highly repetitive sequences (6 – 100 bp, over 10 6 copies in the haploid genome, called satellite DNAs ) represent about 20 % of genome and are clustered in several locations (e.g. at centromers, as telomers). They are not transcribed and exhibit individual specifity so that they may be used for personal identification (DNA fingerprinting). 10 % of the genome are inverted repeats (palindromes with twofold axis of symmetry) and other non-classified (junk) DNA. Human mitochondrial genome has a highly compact structure consisting almost entirely of coding regions with genes for 13 protein subunits, 22 tRNAs and 2 rRNAs.
DNA as a template In DNA replication , both DNA strands act as templates to specify the complementary base sequence on the new chains, by base-pairing. In transcription of DNA into RNA in vivo , only one DNA strand of dsDNA acts as template that is called the negative strand . The base sequence of the transcribed RNA corresponds to that of the coding (positive) strand , except that in RNA thymidine is replaced by uridine. 3´-OH- • • • G T G G A C G A G T C C G G A A T C G • • • -5´-P 5´-P- • • • C A C C T G C T C A G G C C T T A G C • • • -3´-OH 5´-P- • • • C A C C U G C U C A G G C C U U A G C • • • -3´-OH dsDNA transcribed RNA coding strand positive strand template negative strand
Denaturation of DNA Double-stranded DNA is the natural form of DNA. In solutions of dsDNA isolated from the cells, the strands of dsDNA can be separated under certain conditions to obtain solutions of single strands of DNA as random coils – denatured DNA . Most usually, denaturation of dsDNA is called forth by heating the solution to temperature higher than approx. 70 – 80 °C (" melting " of hydrogen bonds between bases). Denatured DNA can be also obtained in high concentration of urea or at extreme pH values. heating heating ssDNA (denatured DNA)
melting temperature T m t / °C absorbance A 260 nm Purine and pyrimidine bases of DNA absorb UV light at 260 nm and the absorbance of dsDNA is lower than that of denatured ssDNA ( hyperchromic effect of DNA denaturation ). DNA double helices that comprise high amounts of guanine and cytosine (there are three hydrogen bonds in those base pairs) undergo denaturation less readily than DNA with high content of adenine and thymine (two H-bonds only). The quantity called melting temperature T m of dsDNA (the temperature at which 50 % of RNA is denatured) is indirectly proportinal to the ratio dA/dG in ds DNA. random coil double helix
Denaturation of DNA is fully reversible , if a hot solution of denatured DNA is cooled slowly , the hydrogen bonds between complementary bases and the original dsDNA are restored – the process is called annealing . If a hot solution is cooled rapidly , hydrogen bonds between bases are formed in a random way. Various either intramolecular or intermolecular aggregates are formed. Hybrid double-stranded helices can be formed between DNAs derived from different molecules or organisms (if the complementary sequences are present in). In solutions containing both RNA and denatured DNA, DNA/RNA hybrids may originate similarly,, if they contain complementary sequence. annealing (slow cooling) rapid cooling intermolecular aggregate
Intercalation of dsDNA Certain types of molecules that have a low molecular mass may slip into a crevice between neighbouring base pairs in dsDNA – the DNA double helix is intercalated by those compounds.. In vitro , intercalation, e.g., by fluorescent compounds, is one of the common techniques for detection of minute amounts of DNA . In vivo , some antibiotics (e.g. actinomycin D, daunorubicin) or platinum(II) complexes intercalate nuclear DNA and cause structural changes that may. disturb the normal course of DNA replication and transcription. Their ability to inhibit the growth of rapidly dividing cells makes them effective chemotherapeutic agents (cytostatics) in the treatment of some cancers.
Human nuclear genome (23 chromosomes, each 1.3 x 10 8 bp) consists of circa 3 x 10 9 bp. There are 23 pairs of chromosomes in diploid cells. In the nuclei, DNA is present in a condensed form as chromatin . Higher levels of DNA organization – chromatin Three higher levels of DNA organization into chromatin: "Bare" d ouble helical DNA 1 st level – fibrils of nucleosomes, 2 nd level – superhelix of nucleosome fibrils , solenoid, 3 rd level – radial loops of solenoids surrounding a central nuclear protein scaffold form the fibres of intermitotic chromatin. In the course of mitosis, chromatin fibres are rearranged into the metaphasic chromosomes .
Fibrils of nucleosomes Histones are basic proteins that comprise about 100 aminoacyl residues, from which approx. 25 % is lysine and arginine. The histone octamers contain molecular types H 2A, H 2B, H 3, and H 4; type H 1 binds on the linker DNA. histone octamer contains two each of histones H 2A,H 2B,H 3, and H 4 histone H 1 the sequences of 60 bp – the linker DNA s with histone H 1 seals off the nucleosome Nucleosomes – two turns of DNA duplex (circa 160 bp) wound around the cluster of histones (octamer) dsDNA (bare double helix, 10 bp per turn) 2 nm 10 nm
(„beads on a thread“) Fibrils of nucleosomes 10 nm Solenoid - fibrils of nucleosomes are coiled in a superhelix 30 nm fibres – diameter 30 nm 1200 bp per turn of the supercoil Fibres of intermitotic chromatin 300 nm 700 nm non-histone proteins radial loops of solenoids (20 000 – 80 000 bp per one loop are anchored to the nuclear protein scaffold Metaphasic chromosomes originate by condensation of intermitotic chromatin fibres
Ribonucleic acids differ from DNA in – having D - ribose , – uridine replaces thymidine, – most molecules are single-stranded , (although helical regions may be present by looping of the chain back on itself – arms and loops). The length of RNA molecules is usually shorter than of DNA chains . Some types of rRNA can function as catalysts in the posttranscriptional processing of primary DNA transcripts. They are called ribozymes and they are able to reassemble RNA strands by cutting off certain sequences and splicing the remainders. - small nuclear RNAs (snRNA) - stable constituents of small nuclear ribonucleoprotein particles (snRNP, " snurps ") - short-lived RNA primers acting in DNA replication - messenger RNAs (mRNA) - ribosomal RNAs (rRNA) - transfer RNAs (tRNA) The major types of RNA
Synthesis of RNA Ribonucleic acids are synthesized through transcription of DNA. In eukaryotic cells, the products of transcription are primary transcripts – the precursors of major RNA types . The precursor RNAs are transformed to functional types in the process called posttranscriptional processing , which includes primarily cutting off certain parts of polynucleotide chains and connecting the remainders (splicing). There are also other post-transcriptional modifications (e.g. modification of purine and pyrimidine bases and adding various groups to both ends of the chains). Most of those changes are provided before the final forms are exported from nuclei to cytoplasm.
Messenger RNA is the carrier of genetic information coded in structural genes. mRNA molecules are the templates for ribosomal proteosynthesis. In eukaryots, one mRNA molecule serves for synthesis of one polypeptide chain – it is the product of one structural gene expression. Precursors of mRNA called hnRNAs (heterogeneous nuclear RNAs) have to undergo posttranscriptional processing, in which the transcripts of non-coding sequences of the gene (introns) are cut off and coding sequences (exons) spliced. Then "cap“ is attached to the 5´-end and a long polyadenylate chain linked to the 3´-end. The mass of mRNA in the cell represents only few percent of the total cellular RNA, but about 10 3 – 10 4 different molecular types may exist. mRNAs have very short biological half-lives (few days on average, the half-lives may differ from each other very much ).
To the 3´-end of mRNA a long poly(A) sequence (about 200 nucleotides) is added. The 5´- methylguanosine "cap" prevents mRNA against 5´-endonucleases and it is also the marker recognized in proteosynthesis. CH 3 GpppNN (A) n A-OH CH 3
Ribosomal RNA is the most abundant RNA (in mass, up to 80 % of total cellular RNA). Only four types of rRNA are present in animal cells: 28 S rRNA, 18 S rRNA, 5.8 S rRNA, and 5 S rRNA. The S is the symbol for Svedberg unit – the sedimentation constant unit that expresses the rate of macromolecule sedimentation measured in an ultracentrifuge. The numerical value is proportional to both the size and shape of macro molecules. M r of rRNA vary from 40 000 to 155 000. Those values correspond with 120 – 4700 nucleotides that comprise the chains. rRNA molecules are constituents of ribosomes : the l arge subunit of ribosome in animals (60 S) contains 28 S rRNA, 5.8 S rRNA, 5 S rRNA, and 49 proteins, the small subunit only one molecule of 18 S rRNA and 33 proteins. Up to 50 % of the polynucleotide chains of rRNA are arranged in helical stem-structures (base-paired " hairpins " of unequal size), many of the bases in rRNA are methylated. rRNA has a relatively very long biological half-live.
Transfer RNA tRNA transfers aminoacyl residues to the ribosomes for peptide bond synthesis. It comprises about 15 % of the total cellular RNA. The molecules are relatively small ( 4 S), each tRNA is a single chain containing about 80 (between 73 and 93) ribonucleotides. In cytoplasm (and in mitochondrial matrix), there is at least one tRNA for each of the 20 amino acids, though not as many as one for each codon. tRNA molecules contain many unusual ( "minority") bases , typically between 7 and 15 per molecule, formed by enzymatic modification of precursor tRNAs; ribosylthymine and pseudouridine are also present. Secondary structure of tRNA About half the nucleotides in tRNAs are base-paired to form four double-helical stems (arms) and three or four loops. tRNA share a common secondary structure that resembles a clover leaf in the two-dimensional drawing. The spatial arrangement of tRNAs (their tertiary structure) takes L- shaped conformation.
Secondary structure of tRNA T ΨC loop binds noncovalently to ribosome surface 5´-end acceptor stem variable loop anticodon arm DHU loop two or more DHU residues at different positions 3´-OH end binds amino acyl through ester bond anticodon 3´-position 3´ 5´ codon of m RNA 5´-wobble position
Amino acids bind to the 3´-OH end of their specific tRNAs cytosine cytosine adenine tRNA ( acceptor stem ) 3´-OH end O NH 2 R–CH–C ester bond
Spatial arrangement of tRNA ( tertiary structure – L-shaped conformation ) 2.5 nm 6 nm acceptor stem anticodon T ΨC loop DHU loop base pairing between T- and D-loops (Hoogsteen geometry)