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5. NUCLEIC ACIDS
Medical Biochemistry Molecular Principles of Structural Organization of Cells
COMPONENTS OF THE NUCLEIC ACIDS
1. NITROGENOUS BASES
The nitrogenous bases are divided into two groups:
1. Purine bases:
Purine Adenine (A, Ade) Guanine (G, Gua)
6-aminopurine 2-amino-6-oxopurine
2. Pyrimidine bases
Pyrimidine Uracil (U, Ura) Thymine (T, Thy) Cytosine (C, Cyt)
2,4-dioxypyrimidine 5-methyluracil 2-oxo-4-aminopyrimidine
N
N NH
N
NH 2
H2N
HN
N NH
N
ON
N NH
N1
2
34
56
7
8
9
N
N1 6
54
3
2
NH
N
NH2
ONH
HN
O
O NH
HN
O
O
CH3
Nitrogenous bases (NB) are classified in:
Major bases
Major purine bases are
• Adenine (A)
was isolated from pancreas and yeast
in both DNA and RNA, in nucleosides mono-, di-, triphosphates, coenzymes
• Guanine (G)
isolated from guano
in both DNA and RNA, nucleosides mono-, di-, triphosphates
Major pyrimidine bases are
• Uracil (U) in RNA, nucleotides, activates the substrates (UDP-glucose), free
• Thymine (T)
isolated from thymus DNA
in DNA, and in small amounts in RNA
• Cytosine (C)
in both DNA and RNA, nucleosides (cytidin-phosphates) acting in the synthesis of phospholipids
Minor bases are primarily found in tRNA and in trace in rRNA; e.g.:
2-methyladenine, 1-methylguanine, 5-methylcytosine, 5-oxymethylcytosine
General properties of the nitrogenous bases
They are hetero-cycles; due to the presence of N have an alkaline character
When H is changed with –OH or –NH2 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 pH<9 (in biological systems) the lactam (keto) form is predominant favoring the formation of covalent bonds of N-glycoside type between N atom in position 1 of pyrimidine or N atom in position 9 of purine and semiacetal –OH (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
NH
HN
O
O N
N
OH
HO
2. PENTOSES -Ribose (R) -2-deoxyribose (dR)
in RNA in DNA
3. PHOSPHORIC ACID MOIETY H3PO4 - PO3H2
It is able to link the nucleotides forming phosphodiester bond between
-OH in position 3’ of the pentose in one nucleotide and
-OH in position 5’ in the other nucleotide
OHPHO
OH
O
CH2-OH
H
OH
H
OH OH
H HO
CH2-OH
H
OH
H
OH H
H HO
OHP
OH
O
Compounds containing nitrogenous base linked to pentose = nucleosides (C-1’ of pentose is linked to N-9 in purine or N-1 in pyrimidine = N- -glycosidic bond)
Ribonucleosides R+ A = adenosine R+ G = guanosine R+ C = cytidine R+ U = uridine
Deoxyribonucleosides dR+A=deoxyadenosine dR+G=deoxyguanosine dR+C=deoxycytidine dR+T=deoxythymidine
NUCLEOSIDES
N
N N
N
NH 2
O
OHOH
HH
H
CH 2
H
HOO
OHOH
HH
H
CH2
H
HO
HN
N N
N
O
H2N
O
OHOH
HH
H
CH2
H
HO
N
N
NH2
O
O
OHOH
HH
H
CH2
H
HO
HN
N
O
O
N
N N
N
NH 2
O
HOH
HH
H
CH 2
H
HOO
HOH
HH
H
CH2
H
HO
HN
N N
N
O
H2N
O
HOH
HH
H
CH2
H
HO
N
N
NH2
O
O
HOH
HH
H
CH2
H
HO
HN
N
O
O
CH3
NUCLEOSIDES
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 tRNA
the most widespread are dihydrouridine, pseudouridine, ribothymidine
NUCLEOTIDES
• 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 + H3PO4 = nitrogenous base + pentose + H3PO4) • The phosphate group can add to positions 2’, 3’, 5’ of ribose 3’, 5’ of deoxyribose adenosine-3’-monophosphate adenosine-5’-monophosphate
• Free nucleotides are nucleosides-5’-P (mononucleotides) that are involved in the synthesis of nucleic acids and are formed by their decomposition
O
OHOH
HH
H
CH2
H
OPHO
OH
O
N
N N
N
NH2
O
OHO
HH
H
CH2
H
PO
OH
HO
OH
N
N N
N
NH2
Ribomononucleotides
R+A+H3PO4 = adenosine-5’-monophosphate = AMP = adenylic acid
R+G+H3PO4 = guanosine-5’-monophosphate = GMP = guanylic acid
R+C+H3PO4 = cytidine-5’-monophosphate = CMP = cytidylic acid
R+U+H3PO4 = uridine-5’-monophosphate = UMP = uridylic acid
Deoxyribomononucleotides
dR+A+H3PO4 = deoxyadenosine-5’-monophosphate = dAMP = deoxyadenylic acid
dR+G+H3PO4 = deoxyguanosine-5’-monophosphate = dGMP = deoxyguanylic acid
dR+C+H3PO4 = deoxycytidine-5’-monophosphate = dCMP = deoxycytidylic acid
dR+T+H3PO4 = deoxythymidine-5’-monophosphate = dTMP = deoxythymidylic acid
O
OHOH
HH
H
CH2
H
OPHO
OH
O
N
N N
N
NH2
O
OH
HH
H
CH2
H
N
N
NH2
O
OH
OP
O
OH
HOO
OHOH
HH
H
CH2
H
HN
N
O
O
OPHO
O
OH
O
HOH
HH
H
CH2
H
OPHO
OH
O
N
N N
N
NH2
O
H
HH
H
CH2
H
N
N
NH2
O
OH
OP
O
OH
HOO
HOH
HH
H
CH2
H
HN
N
O
O
OPHO
O
OH
CH3
O
H
HH
H
CH2
HOH
OPHO
O
OH
HN
N N
N
O
H2N
O
OH
HH
H
CH2
HOH
OPHO
O
OH
HN
N N
N
O
H2N
Nucleosidepolyphosphates 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),
Nucleoside diphosphates and triphosphates are the most frequently occuring
in the cells.
In the cell, all the nucleoside phosphates occur as anions:AMP2-, ADP3-, ATP3-
ADP and ATP are rich in energy (macroergic), used by the organism for
performing different functions.
Other nucleotides are implicated in the function of biological synthesis.
Ribonucleoside phosphates
adenosine-5’-mono-, di-, tri-phosphate = AMP, ADP, ATP
guanosine-5’-mono-, di-, tri-phosphate = GMP, GDP, GTP
cytidine -5’-mono-, di-, tri-phosphate = CMP, CDP, CTP
uridine -5’- mono-,di-, tri-phosphate = UMP, UDP, UTP
AMP ADP ATP
Deoxyribonucleosides phosphates
deoxyadenosine-5’- mono-, di-, tri-phosphate = dAMP, dADP, dATP
deoxyguanosine-5’- mono-, di-, tri-phosphate = dGMP, dGDP, dGTP
deoxycytidine -5’- mono-, di-, tri-phosphate = dCMP, dCDP, dCTP
deoxythymidine-5’- mono-, di-, tri-phosphate = dTMP, dTDP, dTTP
O
OHOH
HH
H
CH2
H
OPHO
OH
O
N
N N
N
NH2
O
OHOH
HH
H
CH2
H
OPO
OH
N
N N
N
NH2
O
P
OH
O
O
P
OH
HO
O
O
OHOH
HH
H
CH2
H
OPO
OH
N
N N
N
NH2
O
P
OH
HO
O
NUCLEOTIDE DERIVATIVES
Cyclic nucleotides (3’,5’-AMPc 3’,5’-GMPc) are universal regulators of intracellular metabolism.
• cAMP
is mediator of the action of hormones as second messenger,
activates and regulates the function of enzymes – allosteric mechanism in metabolic systems.
• cGMP
second messenger for the action of hormones
cAMP cGMP
O
OHO
HH
H
CH2
HPO
OH
O
N
N N
N
NH2
O
OH
HH
H
CH2
HOP
OH
O
HN
N N
N
O
H2N
O
NUCLEOTIDE DERIVATIVES
Nucleotide coenzymes (uridine, cytidine, deoxythymidine, adenosine, guanosine coenzymes) contain residues of saccharides, alcohols, aminoacids, lipids, inorganic compounds:
UDP-glucose (UDPGlc, UDPG) is intermediate in the reversible conversion of glucose in galactose, formation of glycogen in animals or starch in plants.
CDP-choline is involved in the formation of phosphatidyl-choline and choline plasmalogens
CMP-sialic acid
UDP-glucuronic acid is a donor of glucuronic acid residue for the coupling reactions of native or foreign substances
UDP-G CDP-choline
O
OHOH
HH
H
CH2
H
HN
N
O
O
OPO
O
OH
P
OH
OH O
OH OH
OHH
OH
CH2-OH
HH
O
OH
HH
H
CH2
H
N
N
NH2
O
OH
OP
O
OH
OP
OH
O
O
H2CH2CN+
CH3
H3C
CH3
GENERAL PROPERTIES AND BIOCHEMICAL ROLE
OF NUCLEOTIDES
Properties:
• Have an acidic character (the protons in the phosphoric acid moiety dissociate: nucleozid-O-PO3
2-)
• Maximum absorbance at =260nm (UV) due to the presence of nitrogenous bases
• Nucleotides can be hydrolyzed by 5’-nucleotidase, setting the H3PO4 free
Biochemical role:
• In the structure of coenzymes (NAD+, FAD, CoA-SH)
• Coenzymes: UDP-G, CDP-Choline
• Take part in the enzyme catalyzed reactions:
CTP biosynthesis of phospholipids
UTP in biosynthesis and conversion of carbohydrates
• Trinucleotides are precursors in the biosynthesis of nucleic acids
• Second messengers for the hormonal control (3’5’-AMPc, 3’5’-GMPc)
• ATP is the universal macroergic compound of living organisms
Role and biochemical importance of ATP
ATP, ADP, AMP 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: ATP-ase
ATP + H2O ADP + H3PO4
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 ATP represents the storage of chemical energy resulted from the food
2. Transformation of ATP in ADP represents the generation and use of energy stored in the ATP molecule
ATP ADP H3PO4
O
OHOH
HH
H
CH2
H
OPO
OH
N
N N
N
NH2
O
P
OH
O
O
P
OH
HO
O +H2O
-H2O
generation of energy
accumulation of energy
OHPHO
OH
O
O
OHOH
HH
H
CH2
H
OPO
OH
N
N N
N
NH2
O
P
OH
HO
O
POLYNUCLEOTIDES = NUCLEIC ACIDS
Are macromolecular substances result of the condensation of a great number of mononucleotides (structural units)
They are:
• Polyribonucleotides = Ribonucleic acid (RNA)
• Polydeoxyribonucleotides = Deoxyribonucleic acid (DNA)
Distinct characters: DNA RNA
NB: A, G, C, T A, G, C, U
Pentose: dR R
Number of nucleotide monomers DNA > RNA
Length of chain DNA > (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
STRUCTURE AND LEVELS OF ORGANIZATION
OF NUCLEIC ACIDS
PRIMARY STRUCTURE
DNA and RNA are linear polynucleotide chain made up of mononucleotides linked by 3’,5’-phosphodiester bonds: each pentose 3’-OH of one mononucleotide is linked covalently to pentose 5’-OH of the neighboring mononucleotide.
The chains have 2 ends:
5’ end with triphosphate and
3’ end with a free –OH
The chains are polar and directed 5’ 3’ or 3’ 5’ (exception: the circular DNA and RNA of certain viruses and bacteria).
O
HO
HH
H
CH 2
H
OPHO
OH
O
O
H
HH
H
CH 2
HO
O
PHO
O
H
HH
H
CH 2
H
N
N
NH 2
O
O
O
PHO
O
HO
HH
H
CH 2
H
HN
N
O
O
O
P
HO
O
N
N N
N
NH 2
HN
N N
N
O
H2N
CH 3
O
O
5'
5'
5'
5'
3'
3'
3 '
3 '
O
OHO
HH
H
CH 2
H
OPHO
OH
O
O
OH
HH
H
CH 2
HO
O
PHO
O
OH
HH
H
CH 2
H
N
N
NH 2
O
O
O
PHO
O
OHO
HH
H
CH 2
H
HN
N
O
O
O
P
HO
O
N
N N
N
NH 2
HN
N N
N
O
H2N
O
O
5'
5'
5 '
3'
3 '
3'
3 '
5'
Primary structure of DNA Primary structure of RNA
3’.5’-phosphodiester bond
3’.5’-phosphodiester bond
3’.5’-phosphodiester bond
3’.5’-phosphodiester bond
3’.5’-phosphodiester bond
3’.5’-phosphodiester bond A
G
C
T
STRUCTURE AND LEVELS OF ORGANIZATION
OF NUCLEIC ACIDS
PRIMARY STRUCTURE
The genetic text of DNA is composed of
code triplets or codons =
linear sequences of three adjacent nucleotides
The sites of DNA chain that contains information on
the primary structure of all types of RNA are
structural genes.
The order of nucleotides in RNA is the same as that in
the DNA region that is replicated (copied) with the
distinction that RNA consists of ribonucleotides that
contain U instead of T
SECONDARY STRUCTURE
In 1953 Watson and Crick proposed a double-helix model for the DNA 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
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 H bonds
• The interaction of G and C is effected through the
involvement of 3 H bonds
A = T G ≡ C
NN
O
O
CH3
N
NN
N
NH H
H
H
HN
N
N
O
NN
NN
N
OH
H
H
H
H
H
H
H
SECONDARY STRUCTURE OF DNA
Relationship concerning the content of individual bases in DNA
(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 DNA is AT type
(G+C)>(A+T) the DNA is GC type
These rules indicate that the buildup of DNA is effected in a strict
conformity with the pairwise interactions A-T and G-C
TERTIARY STRUCTURE OF DNA
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 DNA, ”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.
TYPES AND LOCATION OF DNA
Nuclear DNA (97-98%) in the chromosomes coupled with
basic proteins (protamines, histones) forming chromatine.
Nucleolus contains associated DNA and RNA
Mitochondrial DNA (1-3%) in the mitochondria matrix
• Structure of simple or double circular helix; does not form
complex with proteins; MW << nuclear DNA
• 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
GENERAL PROPERTIES OF DNA
1. COLLOIDAL BEHAVIOR
On 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. DENATURATION - RENATURATION
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 DNA
results in a separation of its double helix (“helix-coil” transition);
Slowly cooled, the chains reunite according to the
complementarity principle, DNA regaining its native double–
helix; this phenomenon is called renaturation
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 DNA absorbs the UV light maximally at 260nm. The absorption intensity of a native nucleic acid is
increased as the DNA is denatured (hyperchromic effect) or
decreased when the double-helix is reformed (hypochromic effect)
1. HYBRIDIZATION:
the process whereby hybrid duplexes of complementary DNA and RNA combined.
the aptitude of nucleic acid to renaturate after denaturation has provided a valuable method of cloning different genes and other DNA sequences from different organisms
BIOLOGICAL FUNCTIONS OF DNA
The molecular basis of the transmission of genetic information
from one generation to another
Ensures and controls the synthesis of the proteins (enzymes)
In DNA 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
STRUCTURE AND LEVELS OF ORGANIZATION OF
RNA
SECONDARY AND TERTIARY STRUCTURE
Messenger RNA = mRNA
Formed in the cell from pro-mRNA that contains the
transcripts of DNA
The code element of mRNA is a linear sequence of three
adjacent nucleotides = codon or code triplet. Each codon
corresponds to a defined aminoacid.
The secondary structure of mRNA is a bent chain (hairpins
and linear regions)
The tertiary structure is like a thread wound round a spool
(a special transport protein - informofer)
Transfer RNA = tRNA The secondary structure of tRNA 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 tRNA. The 3’ –OH of adenosine is free. At this site the -COOH 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 tRNA = anticodon, complementarily paired to a codon of mRNA; 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 tRNA to the ribosome
4. Dihydrouridine loop (diHU) (8-12 nucleotides) binding aminoacyl-t-RNA synthetase, the enzyme which recognizes the aminoacid
5. Extra loop varies in shape and composition in various tRNA
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
Ribosomal RNA = rRNA
Enters in the structure of the ribosomes.
n ribosomes + 1 mRNA = 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 RNA in nucleus – recognition and activation of DNA genes
Low-molecular RNA in nucleus and cytoplasmic RNA particles – activation of DNA genes formation of the skeleton for protein particles involved in the transfer of RNA from nucleus into the cytoplasm
