PolysaccharidesHeteroglycosides

Nucleosides, nucleotides, nucleic acids

Medical Chemistry 2007 (J.S.)Lecture 10

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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)

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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(14) 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(14) glycosidic bonds are connected at branchpoints by α(16) linkages.

Starch (Lat. amylum)

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Amylose(1→4)--D-glucan (glucopyranan)

the nonreducing end

the free anomeric hydroxyl(the reducing end)

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Amylopectin

Chains with consecutive (1→4) links average only about 30 units in length. Branch points occur every 8 – 12 glucose residues.

-1,6-glycosidic linkageat the branch point

6

1

-1,4-glycosidic linkages

4

6

is the energy-storing polysaccharide in animals.

Like amylopectin of starch, it is made of α(14) and α(16) 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).

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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.

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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 bacteriain 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

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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 andother plant biopolymers (both polysaccharides and lignin) that cannot behydrolyzed 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.

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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)

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Dextran

branching(13)

(16) bonds

is a polysaccharide (unusual D-glucan) produced by some species of bacteriafrom sucrose. The molecular mass of native dextrans is in the range 104 – 106.

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 insolublecomponent of dental plaques that cannot be decomposed by the -amylase of saliva.

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Heteropolysaccharides (heteroglycans)

give two or more different monosaccharide units on completehydrolysis (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

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3) GlcNAc (β1–4) GlcA (β1–3) GlcNAc (β1–4) GlcA(β1

Segment of the hyaluronic acid molecule

The length of hyaluronate linear unbranched chain can reach 4000 nm.

[ N-Ac-glycosamine glycuronic acid ]n

GlycosaminoglycansThe 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).

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Common constituents of glycosaminoglycans:

α-D-glucuronic acid

-D-galacturonic acidN-sulfated glucosamine

-L-iduronic acid(5-epimer of glucuronic acid)

N-Ac-galactosamine 4-sulfate

N-Ac-glucosamine 6-sulfate

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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)

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Hyaluronic acid

(1–4) GlcA (1–3) GlcNAc (1–4)

Chondroitin 6-sulfate

(1–4) GlcA (1–3) GlcNAc-6-sulfate (1–4)

Dermatan 4-sulfate

(1–4) L-IdoA (1–3) GalNAc-4-sulfate (1–4)

Keratan 6-sulfate

(1–3) Gal (1–4) GlcNAc-6-sulfate (1–3)

Typical disaccharide units comprising glycosaminoglycans:

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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-IdoA-2-sulfate (1–4) GlcN-N,6-bissulfate (1–4)

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ProteoglycansIf 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.

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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.

hyaluronate

Monomeric proteoglycan (agrecan)

agrecans

In spite of its large size, core protein of proteoglycans represents only about 5 – 15 % mass of the proteoglycan. The agrecan structure resembles a bottle-brush.

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Oligosaccharides and polysaccharides (homopolysaccharidesas well as heteropolysaccharides) are taken as homoglycosides.

In homopolysaccharides, only saccharide units are bound mutuallythrough 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).

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Examples of naturally occuring (hetero)glycosides:

Salicinglycosylated salicylalcohol

Amygdalinbenzaldehyde, 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.

Nucleosides, nucleotides, nucleic acids

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Purine bases of nucleotides

N

N

N

NH

NH2

adenine6-aminopurine, Ade

N

N

N

NH

OH

H2N

guanine2-amino-6-hydroxypurine, Gua

hypoxanthine6-hydroxypurine

N

N

N

NH

OH

xanthine2,6-dihydroxypurine

N

N

N

NH

HO

OH

N

N

N

NH

1

2

3

4

6

7

8

9

5

purineatypical numbering!

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uracil 2,4-dihydroxypyrimidine, Ura

N

NHO

OH

thymine5-methyluracil, Thy

N

N

CH3

HO

OH

N

N

NH2

HO

cytosine 4-amino-2-hydroxypyrimidine, Cyt

lactim form lactam form of uracilN

N

N

N

O

OH

H

HO

OH

They exist in two tautomeric forms. Only the lactam forms can be ribosylated to give nucleosides:

pyrimidine1,3-diazine

N

N Pyrimidine bases of nucleotides

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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:

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Nucleosides

-N-glycosidic bond

N-base

Nucleosides are heteroglycosides, glycosylated nitrogenous bases.Ribosylated and deoxyribosylated purine and pyrimidine bases arecomponents of nucleotides, substrates of the biosynthesis of nucleic acids.

In most nucleosides, D-ribose or 2-deoxy-D-ribose is attached throughβ-N-glycosidic bond to N9 of purine bases or to N1 of pyrimidine bases.

( )

Systematic names of nucleosides are the, e.g., 9-β-D-ribofuranosylguanine and N1-2'-deoxy-β-D-ribofuranosylthymine,but usually trivial names are preferred.

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Trivial names of nucleosidesare 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 AGuanosine Guo GInosine (base hypoxanthine) Ino I

Xanthosine Xao X

Cytidine Ctd CUridine Urd URibothymidine Thd T

Deoxyribonucleosides

Deoxyadenosine dAdo dADeoxyguanosine dGuo dGDeoxycytidine dCtd dCThymidine dThd dT

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Purine ribonucleosides

xanthosine

guanosine9-β-D-ribofuranosylguanine

inosine9-β-D-ribofuranosylhypoxanthine

adenosine

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Pyrimidine nucleosides

deoxycytidineN1-2'-deoxy-β-D-ribofuranosylcytosine

uridineN1-β-D-ribofuranosyluracil

cytidineN1-β-D-ribofuranosylcytosine

thymidineN1-2'-deoxy-β-D-ribofuranosylthymine

5

pseudouridine ribothymidine

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Synthetic nucleoside analogs

N=N=Nzidovudin

(azidothymidine)aciclovircytarabin

(N1-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 alsouseful in clinical medicine as inhibitors of the biosynthesis of nucleic acids.

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Nucleotides are phosphate esters of nucleosides.Phosphate may be attached through ester bond at carbon atoms5', 3', or (in ribose only) 2'; the primed numbers indicate the positionon 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 Mg2+ and Ca2+.

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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 ofenergy-releasing pathways – ATP and other triphosphatesserve oft in activating of metabolic substrates(phosphorylation, UDP glucose, acyl adenylates, etc),

– have an important role in regulation of metabolism (intracellularconcentrations of ATP and ADP, concentration of cAMP –a second messenger),

– adenine nucleotides are components of various coenzymes(NAD, NADP, FAD, FMN, coenzyme A), etc.

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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

thymidine 3'-phosphate

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Adenosine 5'-monophosphate(AMP, adenylate)

Adenosine 5'-triphosphate (ATP)

+ + H++ H2O

ADP H

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Cyclic GMP (cGMP) has a similar specific function in transduction ofextracellular 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 specificreceptors on the surface of cytoplasmatic membranes. cAMP thenactivates phosphorylation of intracellular proteins, which evokes the cellular respons.

ATP diphoshate + AMP+ H2O

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NAD (nicotinamide adenine dinucleotide) and

NADP (NAD 2'-phosphate) are coenzymes of dehydrogenases:

adenine

anhydride bond

nicotinamide

2'

NAD+ (oxidized form)

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FAD (flavin adenine dinucleotide) andFMN (flavin mononucleotide)

are prosthetic groups of flavin dehydrogenases. Similarly topyridine coenzymes NAD and NADP, they transfer two atoms of hydrogen:

6.7-dimethylisoalloxazine

ribitol(D-ribityl-)

anhydride bond

adenine

coenzyme FAD (oxidized form)

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Coenzyme Ais a transporter of acyls (acetyl, fatty acyls, acyls that are products of2-oxoacids decarboxylation, etc.). Acyls are transported in the formof thioesters:

HO

CH2C

HS CH2 CH2 HN

OC CH2 CH2 HN

OC CH

CH3

CH3

cysteamine pantothenic acid

3'-phosphoADP

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Nucleic acids

are polynucleotides – huge linear polymers consisting of nucleotides joined by 3'→5' phosphodiester bonds.

Deoxyribonucleic acids (DNA) and ribonucleic acids (RNA)are two classes of nucleic acids. Their functions are quite different.

DNAis the hereditary molecule in all cellular life forms as well as inmany (not all) viruses. The section of DNA molecule that encodes(contains information for the synthesis of) a unique functional proteinis 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.

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RNAhas 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.)

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3'→5' phosphodiester linkbetween 3'-OH and 5'-OH

Polynucleotide chain structure

5'-phosphateend

free 3'-OH end(event. 3'-phosphate end)

Bases are attached to the sugar-phosphate backbone throughβ-N-glycosidic bonds.

By convention, direction ofreading is from 5'-end to 3'-end.

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P P5'-P

P P P 3'-OH

Ura Cyt Ade Ade Gua CytRNA chain

Symbolic notation of the base sequence

From the 5'-end: pU→C→A→A→G→C

pUpCpApApGpC pU-C-A-A-G-C Identical chain from the 3'-end:

5'-UCAAGC C←G←A←A←C←Up

UCAAGC 3'-CGAACU

Primary structure - abbreviated notation

DNA chain pdG-dC-dT-dT-dG-dA d(pGCTTGA) or d(A←G←T←T←C←G) GCTTGA 3'-AGTTCG

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Deoxyribonucleic acid

One dsDNA molecule (one human haploid chromosome, chromatide), consists of (1 – 3) 108 base pairs (bp), the average value is 1.3 108 bp. The average relative molecular mass Mr of two nucleotides equals 660, so that the molecular mass Mr of a dsDNA molecule can reach 1011.

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 only3 % 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.

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2 April 1953J. 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 toJ.D. Watson, F.H.C. Crick, M.H.F. Wilkins

“ ...for their discoveries concerning the molecular structure of nucleic acids andits significance for information transfer in living material…”

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Two polynucleotide chains wind about a common axis with a right-handed twist. Two strands are antiparallel, they run in opposite direction.The coiling is plectonemic – the strands cannot be

separated without unwinding the helix.

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.

5´-end 3´-end

5´-end3´-end

the main helical axis

Secondary double helical structure of DNA

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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.

sugar-phosphate backbone

Top view:

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Pairing of bases (Watson-Crick geometry)is the principle of chain complementarity:

dGdC(G≡C in RNAs)

dA=dT(A=U in RNAs)

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 Guaequals 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

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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.

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34 nm

3.4 nm

5´-end

3´-end

3´-end5´-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 pairsper one turn

Two unequal grooves arise on the surface because the glycosidic bonds of a base pair are not diametrically opposite each other.

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Grooves are the sites of specific binding of regulatory molecules

Phosphate groups on the grooves edgescan 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

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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 hybridsand 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.

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Z-DNA

is a left-handed double helix that has 12 Watson-Crick base pairsper 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-

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Comparison of DNA conformational forms

B-formA-form Z-form

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C(2´)-endo for PyC(3´)-endo for Pu

C(3´)-endoC(2´)-endoSugar pucker

narrow and deepbroad and shallownarrow and

deepMinor groove

flatnarrow and deepwide and deepMajor groove

720°1º - 6ºBase tilt from normal to the helix axis

45 nm28 nm34 nmHelical pitch(rise per turn)

30°33°36ºHelical twist per bp

121110Base pairs perhelical turn

18 nm26 nm20 nmDiameter

left-handedright handedright-handedHelical sense

dsDNA B form A form Z form

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Human nuclear genomeconsists of circa 3 109 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 106 copies) and highly repetitive sequences (6 – 100 bp, over 106 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 genomehas a highly compact structure consisting almost entirely of coding regions with genes for 13 protein subunits, 22 tRNAs and 2 rRNAs.

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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 dsDNAacts as template that is called the negative strand. The base sequence ofthe 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 strandpositive strand

templatenegative strand

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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 thesolution 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)

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melting temperature Tm

t / °C

absorbance A260 nm

Purine and pyrimidine bases of DNA absorb UV light at 260 nm andthe 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 Tm 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

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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 areformed 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

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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.

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Human nuclear genome (23 chromosomes, each 1.3 x 108 bp) consists of circa 3 x 109 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" double helical DNA

1st level – fibrils of nucleosomes,

2nd level – superhelix of nucleosome fibrils, solenoid,

3rd 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 themetaphasic chromosomes.

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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 eachof histones H 2A,H 2B,H 3, and H 4

histone H 1

the sequences of 60 bp – the linker DNAs 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

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(„beads on a thread“)

Fibrils of nucleosomes 10 nm

Solenoid - fibrils of nucleosomes are coiled in a superhelix

30 nm

fibres – diameter 30 nm1200 bp per turn of the supercoil

Fibres of intermitotic chromatin

300 nm700 nm

non-histone proteins

radial loops of solenoids(20 000 – 80 000 bp perone loop are anchored tothe nuclear protein scaffold

Metaphasic chromosomesoriginate by condensation ofintermitotic chromatin fibres

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Ribonucleic acidsdiffer 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

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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.

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Messenger RNA

is the carrier of genetic information coded in structural genes. mRNAmolecules 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 codingsequences (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 103 – 104 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 ).

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To the 3´-end of mRNA a longpoly(A) sequence (about 200nucleotides) is added.

The 5´-methylguanosine "cap"prevents mRNA against5´-endonucleases and it isalso the marker recognized inproteosynthesis.

CH3

GpppNN (A)nA-OH

CH3

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Ribosomal RNAis 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 thatexpresses the rate of macromolecule sedimentation measured in an ultracentrifuge.The numerical value is proportional to both the size and shape of macromolecules.

Mr of rRNA vary from 40 000 to 155 000. Those values correspondwith 120 – 4700 nucleotides that comprise the chains.

rRNA molecules are constituents of ribosomes: the large 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 arrangedin helical stem-structures (base-paired "hairpins" of unequal size), manyof the bases in rRNA are methylated.rRNA has a relatively very long biological half-live.

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Transfer RNAtRNA 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 tRNAfor 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.

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TΨC loopbinds noncovalentlyto ribosome surface

5´-end

acceptor stem

variable loop

anticodon arm

DHU looptwo or more DHU residues

at different positions

3´-OH end binds amino acyl through ester bond

anticodon3´-position

3´ 5´codon of mRNA

5´-wobble position

Secondary structure of tRNA

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Amino acids bind to the 3´-OH end of their specific tRNAs

cytosine

cytosine

adenine

tRNA (acceptor stem)

3´-OH end

ONH2

R–CH–C

ester bond

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Spatial arrangement of tRNA(tertiary structure – L-shaped conformation)

2.5 nm

6 nm

acceptor stem

anticodon

TΨC loop

DHU loop

base pairing betweenT- and D-loops(Hoogsteen geometry)