Medical Biochemistry Molecular Principles of Structural Organization of Cells 5. NUCLEIC ACIDS

Transcription

1 Medical Biochemistry Molecular rinciples of Structural rganization of Cells 5. UCLEIC ACIDS

2 CMETS F TE UCLEIC ACIDS 1. ITRGEUS BASES The nitrogenous bases are divided into two groups: 1. urine bases: 1 2 urine Adenine (A, Ade) Guanine (G, Gua) 6-aminopurine 2-amino-6-oxopurine 2. yrimidine bases yrimidine Uracil (U, Ura) Thymine (T, Thy) Cytosine (C, Cyt) 2,4-dioxypyrimidine 5-methyluracil 2-oxo aminopyrimidine C 3 2

3 itrogenous bases (B) are classified in: Major bases Major purine bases are Adenine (A) was isolated from pancreas and yeast in both DA and RA, in nucleosides mono-, di-, triphosphates, coenzymes Guanine (G) isolated from guano in both DA and RA, nucleosides mono-, di-, triphosphates Major pyrimidine bases are Uracil (U) in RA, nucleotides, activates the substrates (UD-glucose), free Thymine (T) isolated from thymus DA in DA, and in small amounts in RA Cytosine (C) in both DA and RA, nucleosides (cytidin-phosphates) acting in the synthesis of phospholipids Minor bases are primarily found in tra and in trace in rra; e.g.: 2-methyladenine, 1-methylguanine, 5-methylcytosine, 5-oxymethylcytosine

4 General properties of the nitrogenous bases They are hetero-cycles; due to the presence of have an alkaline character When is changed with or 2 the solubility in water is reduced, the melting point increases The bases have the ability to undergo a lactam-lactim (keto-enol) tautomerism The amine compounds have an alkaline character, the enol compounds act as acids. At p<9 (in biological systems) the lactam (keto) form is predominant favoring the formation of covalent bonds of -glycoside type between atom in position 1 of pyrimidine or atom in position 9 of purine and semiacetal (C-1) of pentose They have a maximum absorbance at =260nm (UV) used to dose with spectrophotometric method in UV Low solubility in cold water; soluble in alkaline solutions

5 2. ETSES -Ribose (R) -2-deoxyribose (dr) C 2 - C 2 - in RA in DA 3. SRIC ACID MIETY It is able to link the nucleotides forming phosphodiester bond between - in position 3 of the pentose in one nucleotide and - in position 5 in the other nucleotide

6 UCLESIDES Compounds containing nitrogenous base linked to pentose = nucleosides (C-1 of pentose is linked to -9 in purine or -1 in pyrimidine = - -glycosidic bond) Ribonucleosides R+ A = adenosine R+ G = guanosine R+ C = cytidine R+ U = uridine 2 2 C 2 2 C 2 C 2 C 2 Deoxyribonucleosides dr+a=deoxyadenosine dr+g=deoxyguanosine dr+c=deoxycytidine dr+t=deoxythymidine 2 C 2 2 C 2 C 2 2 C 2 C 3

7 UCLESIDES Are considered products of the partial hydrolysis of nucleotides Ribonucleosides exist free in small amounts Deoxyribonucleosides do not exist free Minor nucleosides contain minor nitrogenous bases exist in tra the most widespread are dihydrouridine, pseudouridine, ribothymidine

8 UCLETIDES They are the monomer units of the nucleic acids; they result from the partial hydrolysis of the nucleic acids under the action of nucleases They are phosphoric esters of the nucleosides (nucleotide = nucleoside = nitrogenous base + pentose ) The phosphate group can add to positions 2, 3, 5 of ribose 3, 5 of deoxyribose adenosine-3 -monophosphate C 2 2 C 2 adenosine-5 -monophosphate Free nucleotides are nucleosides-5 - (mononucleotides) that are involved in the synthesis of nucleic acids and are formed by their decomposition 2

9 Ribomononucleotides R+A+ 3 4 = adenosine-5 -monophosphate = AM = adenylic acid R+G+ 3 4 = guanosine-5 -monophosphate = GM = guanylic acid R+C+ 3 4 = cytidine-5 -monophosphate = CM = cytidylic acid R+U+ 3 4 = uridine-5 -monophosphate = UM = uridylic acid 2 2 C 2 2 C 2 C 2 C 2 Deoxyribomononucleotides dr+a+ 3 4 = deoxyadenosine-5 -monophosphate = dam = deoxyadenylic acid dr+g+ 3 4 = deoxyguanosine-5 -monophosphate = dgm = deoxyguanylic acid dr+c+ 3 4 = deoxycytidine-5 -monophosphate = dcm = deoxycytidylic acid dr+t+ 3 4 = deoxythymidine-5 -monophosphate = dtm = deoxythymidylic acid 2 2 C 3 C 2 2 C 2 C 2 C 2

10 ucleosidepolyphosphates are formed by linking an additional phosphate group. The nucleotides may contain 1 phosphoric acid moiety - mononucleotides (monophosphate nucleosides), 2 phosphoric acid moieties - dinucleotides (diphosphate nucleosides), 3 phosphoric acid moieties - trinucleotides (triphosphate nucleosides), ucleoside diphosphates and triphosphates are the most frequently occuring in the cells. In the cell, all the nucleoside phosphates occur as anions:am 2-, AD 3-, AT 3- AD and AT are rich in energy (macroergic), used by the organism for performing different functions. ther nucleotides are implicated in the function of biological synthesis.

11 Ribonucleoside phosphates adenosine-5 -mono-, di-, tri-phosphate = AM, AD, AT guanosine-5 -mono-, di-, tri-phosphate = GM, GD, GT cytidine -5 -mono-, di-, tri-phosphate = CM, CD, CT uridine -5 - mono-,di-, tri-phosphate = UM, UD, UT C 2 C 2 AM AD AT C 2 Deoxyribonucleosides phosphates deoxyadenosine-5 - mono-, di-, tri-phosphate deoxyguanosine-5 - mono-, di-, tri-phosphate deoxycytidine -5 - mono-, di-, tri-phosphate deoxythymidine-5 - mono-, di-, tri-phosphate = dam, dad, dat = dgm, dgd, dgt = dcm, dcd, dct = dtm, dtd, dtt

12 cam cgm UCLETIDE DERIVATIVES Cyclic nucleotides (3,5 -AM c 3,5 -GM c ) are universal regulators of intracellular metabolism. cam is mediator of the action of hormones as second messenger, activates and regulates the function of enzymes allosteric mechanism in metabolic systems. cgm second messenger for the action of hormones 2 2 C 2 C 2

13 UCLETIDE DERIVATIVES ucleotide coenzymes (uridine, cytidine, deoxythymidine, adenosine, guanosine coenzymes) contain residues of saccharides, alcohols, aminoacids, lipids, inorganic compounds: UD-glucose (UDGlc, UDG) is intermediate in the reversible conversion of glucose in galactose, formation of glycogen in animals or starch in plants. CD-choline is involved in the formation of phosphatidyl-choline and choline plasmalogens CM-sialic acid UD-glucuronic acid is a donor of glucuronic acid residue for the coupling reactions of native or foreign substances 2 C 2 - C 2 3 C C C C 3 2 C C 2 UD-G CD-choline

14 GEERAL RERTIES AD BICEMICAL RLE F UCLETIDES roperties: ave an acidic character (the protons in the phosphoric acid moiety dissociate: nucleozid ) Maximum absorbance at =260nm (UV) due to the presence of nitrogenous bases ucleotides can be hydrolyzed by 5 -nucleotidase, setting the 3 4 free Biochemical role: In the structure of coenzymes (AD +, FAD, CoA-S) Coenzymes: UD-G, CD-Choline Take part in the enzyme catalyzed reactions: CT biosynthesis of phospholipids UT in biosynthesis and conversion of carbohydrates Trinucleotides are precursors in the biosynthesis of nucleic acids Second messengers for the hormonal control (3 5 -AMc, 3 5 -GMc) AT is the universal macroergic compound of living organisms

15 Role and biochemical importance of AT AT, AD, AM take part in processes of storage and utilization of the energy set free during the cellular metabolism They act as donors or acceptors of phosphate moiety The reaction: AT-ase AT + 2 AD reflects the energy flow in the cell; it provides the transfer of the chemical energy used in the cellular metabolism. This process implies 2 fundamental aspects: 1. Formation of AT represents the storage of chemical energy resulted from the food 2. Transformation of AT in AD represents the generation and use of energy stored in the AT molecule 2 C 2 generation of energy +2-2 accumulation of energy 2 C 2 AT AD 3 4

16 LYUCLETIDES = UCLEIC ACIDS Are macromolecular substances result of the condensation of a great number of mononucleotides (structural units) They are: olyribonucleotides = Ribonucleic acid (RA) olydeoxyribonucleotides = Deoxyribonucleic acid (DA) Distinct characters: DA RA B: A, G, C, T A, G, C, U entose: dr R umber of nucleotide monomers DA > RA Length of chain DA > (except some viruses) Structure double helix 1 chain Due to the acidic character, nucleic acids are linked with basic proteins, (histones and protamines) and neutal proteins forming deoxyribo-nucleoproteins ribonucleoprotein

17 STRUCTURE AD LEVELS F RGAIZATI F UCLEIC ACIDS RIMARY STRUCTURE DA and RA are linear polynucleotide chain made up of mononucleotides linked by 3,5 -phosphodiester bonds: each pentose 3 - of one mononucleotide is linked covalently to pentose 5 - of the neighboring mononucleotide. The chains have 2 ends: 5 end with triphosphate and 3 end with a free The chains are polar and directed 5 3 or 3 5 (exception: the circular DA and RA of certain viruses and bacteria).

18 phosphodiester bond 3.5 -phosphodiester bond 3.5 -phosphodiester bond 5' C 2 3' 2 5' C 2 3' 5' C 2 3' G A 2 C 3 5' C 2 3' 5' C 2 3' 3.5 -phosphodiester bond 2 5' C 2 3' C 3.5 -phosphodiester bond T 3.5 -phosphodiester bond 5' C 2 3' 2 5' C 2 3' rimary structure of DA rimary structure of RA

19 STRUCTURE AD LEVELS F RGAIZATI F UCLEIC ACIDS RIMARY STRUCTURE The genetic text of DA is composed of code triplets or codons = linear sequences of three adjacent nucleotides The sites of DA chain that contains information on the primary structure of all types of RA are structural genes. The order of nucleotides in RA is the same as that in the DA region that is replicated (copied) with the distinction that RA consists of ribonucleotides that contain U instead of T

20 SECDARY STRUCTURE In 1953 Watson and Crick proposed a double-helix model for the DA secondary structure The chains are directed antiparallelly (one chain runs in 5 3 direction and the second 3 5 direction) The pentose phosphate moieties are directed outwards The bases protrude into the interior of the helix

21 Formed by specific pairing of a base of one polynucleotide chain with a base of the other chain. The correspondence of the base pairs is called complementarity The interaction of A and T is effected through the involvement of 2 bonds The interaction of G and C is effected through the involvement of 3 bonds C 3 A = T G C

22 SECDARY STRUCTURE F DA Relationship concerning the content of individual bases in DA (Chargaff, 1949): 1. A+G = C+T or (A+G)/(C+T) = 1 2. A = T or A/T = 1 3. G = C or G/C =1 4. A+C = G+T or 6-amino group = 6-keto group 5. (A+T) and (G+C) are the only variable; if: (A+T)>(G+C) the DA is AT type (G+C)>(A+T) the DA is GC type These rules indicate that the buildup of DA is effected in a strict conformity with the pairwise interactions A-T and G-C

23 TERTIARY STRUCTURE F DA The double helical molecule is twisted looking like a supercoil or a bent double-helix It has a great flexibility; the conformation is not rigid. There are differences between the native DA, in vivo, and the one in vitro ; by removing the water and dependent on the electrolytes in the environment, the double-helix is structurally altered.

24 TYES AD LCATI F DA uclear DA (97-98%) in the chromosomes coupled with basic proteins (protamines, histones) forming chromatine. ucleolus contains associated DA and RA Mitochondrial DA (1-3%) in the mitochondria matrix Structure of simple or double circular helix; does not form complex with proteins; MW << nuclear DA Function: Takes part in maintenance of the mitochondria structure Contains the information necessary to synthesize specific proteins intra and extra mitochondria May control the synthesis of the ribosomes Site of the genetic mutations

25 GEERAL RERTIES F DA 1. CLLIDAL BEAVIR n dissolution, nucleic acids become swollen and form viscous, colloid-like solutions; the hydrophilicity is mainly determined by the occurrence of phosphate moieties; in solution the nucleic acids exist as polyanions with acidic properties. Double-stranded nucleic acids are less soluble than single-stranded ones 1. DEATURATI - REATURATI Is produced by heating and the action of chemical agents which break hydrogen and van der Waals bonds stabilizing the secondary and tertiary structures. E.g.: heating DA results in a separation of its double helix ( helix-coil transition); Slowly cooled, the chains reunite according to the complementarity principle, DA regaining its native double helix; this phenomenon is called renaturation

26 The helical structure rotate the plane of polarized light exhibiting an optical activity while the breakdown of the spatial arrangement reduce the optical activity to zero. The DA absorbs the UV light maximally at 260nm. The absorption intensity of a native nucleic acid is increased as the DA is denatured (hyperchromic effect) or decreased when the double-helix is reformed (hypochromic effect) 1. YBRIDIZATI: the process whereby hybrid duplexes of complementary DA and RA combined. the aptitude of nucleic acid to renaturate after denaturation has provided a valuable method of cloning different genes and other DA sequences from different organisms

27 BILGICAL FUCTIS F DA The molecular basis of the transmission of genetic information from one generation to another Ensures and controls the synthesis of the proteins (enzymes) In DA there is encoded the genetic program of development, maintenance and reproduction of each organism Ensures the differentiation and regulation of cells and the constance of the cell replication Is the molecular basis of the natural or induced genetic mutations

28 STRUCTURE AD LEVELS F RGAIZATI F RA SECDARY AD TERTIARY STRUCTURE Messenger RA = mra Formed in the cell from pro-mra that contains the transcripts of DA The code element of mra is a linear sequence of three adjacent nucleotides = codon or code triplet. Each codon corresponds to a defined aminoacid. The secondary structure of mra is a bent chain (hairpins and linear regions) The tertiary structure is like a thread wound round a spool (a special transport protein - informofer)

29 Transfer RA = tra The secondary structure of tra is a shape of clover-leaf determined by intrachain pairing of complementary nucleotides in certain regions of the chain: 1. Acceptor region (end or terminus) - 4 linearly linked nucleotides of which CCA sequence is common in all types of tra. The 3 of adenosine is free. At this site the -C of the aminoacid is added to be transported to the ribosomes, to be used in the protein synthesis. 2. Anticodon loop (7 nucleotides) contains a triplet specific for each tra = anticodon, complementarily paired to a codon of mra; the interaction betweencodon and anti-codon determines the order of the aminoacids in the polypeptide chain 3. Thymine-pseudouracil (TΨC) loop (7 nucleotides) involved in binding the tra to the ribosome 4. Dihydrouridine loop (diu) (8-12 nucleotides) binding aminoacyl-t-ra synthetase, the enzyme which recognizes the aminoacid 5. Extra loop varies in shape and composition in various tra The tertiary structure shape of a bent elbow; the cloverleaf loops are folded back on the molecular framework and held together by Van der Waals bonds

30 Ribosomal RA = rra Enters in the structure of the ribosomes. n ribosomes + 1 mra = polisome Secondary structure: helical regions alternating with nonhelical bent regions Tertiary structure constitutes the framework for the ribosome; ribosomes proteins adhere to the tertiary structure on the outside. Chromosomal RA in nucleus recognition and activation of DA genes Low-molecular RA in nucleus and cytoplasmic RA particles activation of DA genes formation of the skeleton for protein particles involved in the transfer of RA from nucleus into the cytoplasm