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Medical Biochemistry
Molecular Principles of Structural Organization of Cells
5. NUCLEIC ACIDS
COMPONENTS OF THE NUCLEIC ACIDS
1. NITROGENOUS BASES
The nitrogenous bases are divided into two groups:
1. Purine bases:
Purine
N
6
1
5
2
4
N
Adenine (A, Ade)
Guanine (G, Gua)
6-aminopurine
NH2
2-amino-6-oxopurine
8
3
O
7
9
N
H
N
N
N
N
H
N
N
HN
H2N
N
N
H
2. Pyrimidine bases
Pyrimidine
Uracil (U, Ura)
2,4-dioxypyrimidine
Thymine (T, Thy)
5-methyluracil
O
N
4
3
1
N
2-oxo-4-aminopyrimidine
NH2
O
CH3
5
HN
2
Cytosine (C, Cyt)
HN
N
6
O
N
H
O
N
H
O
N
H
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
O
OH
HN
O
N
N
H
HO
N
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
2. PENTOSES
-Ribose (R)
CH2-OH
H
-2-deoxyribose (dR)
OH
O
H
OH
in RNA
OH
O
H
H
H
OH
CH2-OH
H
H
H
OH
H
in DNA
3. PHOSPHORIC ACID MOIETY
H3PO4
O
HO P OH
OH
-
- PO3H2
O
P OH
OH
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
NUCLEOSIDES
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
N
N
N
N
CH2
H
N
HN
H2N
H
H
OH
H
OH
H
O
N
O
N
N
HO CH2
O
R+ U = uridine
NH2
O
NH2
HO
R+ C = cytidine
HN
HO CH2 O
H
H
O
H
H
OH
H
OH
H
OH
O
N
HO
H
OH
CH2
H
N
O
H
H
OH
H
OH
Deoxyribonucleosides
dR+A=deoxyadenosine dR+G=deoxyguanosine
N
N
N
N
CH2
H
H
H
OH
H
H
H
O
N
N
HO CH2
O
O
H
H
OH
H
H
H
OH
O
N
HO CH2 O
H
H
O
H
CH3
HN
N
N
HN
H2N
dR+T=deoxythymidine
NH2
O
NH2
HO
dR+C=deoxycytidine
HO
H
CH2
H
N
O
H
H
OH
H
H
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
NH2
NH2
N
N
N
N
HO CH2 O
H
H
H
H
O
OH
adenosine-3’-monophosphate
O
N
N
P
OH
OH
O
HO P
OH
N
O
N
CH2 O
H
H
H
H
OH OH
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
Ribomononucleotides
R+A+H3PO4
= adenosine-5’-monophosphate
R+G+H3PO4
= guanosine-5’-monophosphate
R+C+H3PO4
= cytidine-5’-monophosphate
R+U+H3PO4
= uridine-5’-monophosphate
N
O
HO P
N
O
OH
N
CH2 O
H
H
H
H
OH OH
N
HN
H2N
OH
HO P
O
N
N
CH2
OH
H
O
O
HO P
O
H
H
OH
H
OH
O
HO P
OH
N
O
N
O CH2
OH
H
CH2 O
H
H
H
H
OH H
HO P
O
HO P
O
N
N
H
OH
H
OH
= dAMP
= dGMP
= dCMP
= dTMP
H
H
N
O
H
H
OH
H
OH
= deoxyadenylic acid
= deoxyguanylic acid
= deoxycytidylic acid
= deoxythymidylic acid
OH
H
H
O
O
O CH2
OH
H
O
N
HO P
O
H
OH
O CH2
OH
H
N
CH2
O
O
NH2
HN
H2N
OH
HN
N
O
N
N
O
N
Deoxyribomononucleotides
dR+A+H3PO4
= deoxyadenosine-5’-monophosphate
dR+G+H3PO4
= deoxyguanosine-5’-monophosphate
dR+C+H3PO4
= deoxycytidine-5’-monophosphate
dR+T+H3PO4
= deoxythymidine-5’-monophosphate
NH2
= adenylic acid
= guanylic acid
= cytidylic acid
= uridylic acid
NH2
O
NH2
N
= AMP
= GMP
= CMP
= UMP
H
N
H
OH
H
O
O
HO P
O
H
CH3
HN
O CH2
OH
H
H
N
O
H
H
OH
H
H
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-, ATP3ADP 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
guanosine-5’-mono-, di-, tri-phosphate
cytidine -5’-mono-, di-, tri-phosphate
uridine -5’- mono-,di-, tri-phosphate
NH2
O
HO P
N
O
OH
AMP
NH2
NH2
N
N
= AMP, ADP, ATP
= GMP, GDP, GTP
= CMP, CDP, CTP
= UMP, UDP, UTP
N
CH2 O
H
H
H
H
OH OH
N
N
O
O
HO P O
OH
N
O
O CH2 O
H
H
OH
H
H
OH OH
HO P
P
N
N
N
ADP
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
O
O
OH
P
OH
O
O
N
N
P
O CH2 O
H
H
OH
H
H
OH OH
ATP
= dAMP, dADP, dATP
= dGMP, dGDP, dGTP
= dCMP, dCDP, dCTP
= dTMP, dTDP, dTTP
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
O
NH2
N
N
N
N
O
O
P
CH2
H
O
H
O
H
OH
OH
cAMP
H2 N
O
H
N
HN
O
CH2
H
P
N
N
O
H
H
O
H
OH
OH
cGMP
NUCLEOTIDE DERIVATIVES
Nucleotide coenzymes (uridine, cytidine, deoxythymidine, adenosine,
guanosine coenzymes) contain residues of glucides, 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 radical for the coupling
reactions of native or foreign substances
O
NH2
HN
H
CH2-OH
O
H
H
OH
H
O
OH
H
OH
O
P
OH
O
O
O
P O CH2
OH
H
UDP-G
N
N
CH3
O
+
H3C N H2C H2C O P
O
H
H
OH
H
OH
CH3
OH
O
O P O
O
CH2
OH
H
CDP-choline
N
O
H
H
OH
H
OH
GENERAL PROPERTIES AND BIOCHEMICAL ROLE
OF NUCLEOTIDES
Properties:
– Have an acidic character (the protons in the phosphoric acid moiety
dissociate: nucleozid-O-PO32-)
– 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
NH2
N
N
O
HO P
OH
O
O
P
+H2O
O
O CH2 O
H
H
OH
H
H
OH OH
-H2O
OH
ATP
P
N
N
N
N
O
NH2
generation of energy
accumulation of energy
O
HO P O
OH
O
P
N
N
O CH2 O
H
H
OH
H
H
OH OH
ADP
O
HO P OH
OH
H3PO4
POLYNUCLEOTIDES = NUCLEIC ACIDS
Are macromolecular substances result of the condensation of a great
number of mononucleotides (structural units)
They are:
– Polyribonucleotides
– Polydeoxyribonucleotides
Distinct characters:
NB:
Pentose:
Number of nucleotide monomers
Length of chain
Structure
= Ribonucleic acid (RNA)
= Deoxyribonucleic acid (DNA)
DNA
A, G, C, T
dR
DNA
DNA
double helix
RNA
>
>
A, G, C, U
R
RNA
(except some viruses)
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).
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
NH2
NH2
N
N
O
5'
N
N
N
O
N
N
N
5'
HO P
HO P
O CH2 O
H 3'
H
OH
O
H
H
O
H
HN
O
3’.5’-phosphodiester
P H2N
N
bond
HO
O
OH
N
G
3’.5’-phosphodiester bond
N
O
HO
CH2 O
H 3'
H
O
H
H
O
OH
HN
P
H2N
5'
CH2
H
O
O
H 3'
H
O
H
NH2
H
N
A
O
3’.5’-phosphodiester bond
N
N
5'
O
N
P
O
HO
H
C
3’.5’-phosphodiester bond
N
CH2
H
O
H
H
P
HO
P
CH3
HN
O
N
CH2
H
Primary structure of DNA
T
N
O
CH2
H
3’.5’-phosphodiester bond
O
H 3'
H
O
H
OH
H3'
H
O
H
O
HN
O
P
O
N
5'
O
O
N
5'
HO
5'
O
H
OH
O
O
3’.5’-phosphodiester bond
O
O
H
NH2
H
O
O
H 3'
O
H 3'
HO
5'
O
CH2
CH2
H
H
Primary structure of RNA
O
H3'
H
O
H
OH
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
H
CH3
O
H
N
N
N
H
N
N
A=T
H
H
H
H
H N
N
O
N
N
O
N
N
H
N
H
N
H
O
N
H
G≡C
H
N
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.
2.
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
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.


3.
The DNA absorbs the UV light maximally at 260nm. The
absorption intensity of a native nucleic acid is
increased as the DNA is denaturated (hyperchromic effect)
or
decreased when the double-helix is reformed (hypochromic
effect)
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
1.
2.
3.
4.
5.
The secondary structure of tRNA is a shape of clover-leaf determined by
intrachain pairing of complementary nucleotides in certain regions of the
chain:
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.
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
Thymine-pseudouracil (TΨC) loop (7 nucleotides) involved in binding the
tRNA to the ribosome
Dihydrouridine loop (diHU) (8-12 nucleotides) binding aminoacyl-t-RNA
synthetase, the enzyme which recognizes the aminoacid
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