BIO 203 Biochemistry I
by
Seyhun YURDUGÜL,Ph.D.
Lecture 8
Nucleic Acids
Content outline
• Nucleosides and nucleotides
• Synthetic nucleotide analogues
• Polynucleotides
• Thermal properties of DNA
• Electrophoresis
Introduction
– Nucleotides: one of the most important
metabolites of the cell.
– found primarily as the monomeric units
comprising the major nucleic acids of the cell,
RNA and DNA.
– also are required for numerous other important
functions within the cell.
These functions include:
• 1. serving as energy stores for future use in
phosphate transfer reactions.
• These reactions are predominantly carried
out by ATP.
• 2. forming a portion of several important
coenzymes:
• such as NAD+, NADP+, FAD and coenzyme
A.
NAD (Nicotinamide adenin dinucleotide)
These functions include:
• 3. serving as mediators of numerous important
cellular processes:
• such as second messengers in signal transduction
events.
• The predominant second messenger:
• Cyclic AMP (cAMP), a cyclic derivative of AMP
formed from ATP.
• 4. controlling numerous enzymatic reactions
through allosteric effects on enzyme activity.
These functions include:
• 5. serving as activated intermediates in
numerous biosynthetic reactions:
• include S-adenosylmethionine (S-AdoMet)
involved in methyl transfer reactions,
• as well as the many sugar coupled
nucleotides involved in:
• glycogen and glycoprotein synthesis.
Nucleoside and nucleotide
structure and nomenclature:
• derivatives of the heterocyclic highly basic,
compounds:
• purine and pyrimidine.
Purine Pyrimidine
Structure
• It is the chemical basicity of the nucleotides
that has given them the common term
"bases"
• as they are associated with nucleotides
present in DNA and RNA.
Structure
• In cells: Five major bases.
• The derivatives of purine are called adenine
and guanine,
• and the derivatives of pyrimidine are called
thymine, cytosine and uracil.
• The common abbreviations used for these
five bases are, A, G, T, C and U.
Base Formulas (Base
substitution:X=H)
Thymine, T
Cytosine, C
Adenine, A
Uracil, U
Guanine, G
Nucleoside forms
• If X=ribose or deoxyribose
• Cytidine
• Uridine
• Thymidine
• Adenosine
• Guanosine
Nucleotide forms
• If X=ribose phosphate
• Cytidine monophosphate (CMP)
• Uridine monophosphate (UMP)
• Thymidine monophosphate (TMP)
• Adenosine monophosphate (AMP)
• Guanosine monophosphate (GMP)
Nucleosides
• The purine and pyrimidine bases in cells:
• linked to carbohydrate and in this form:
• termed as ‘nucleosides’.
• The nucleosides are coupled to D-ribose or 2'-
deoxy-D-ribose;
• through a β-N-glycosidic bond:
• between the anomeric carbon of the ribose
• and the N9 of a purine or N1 of a pyrimidine.
Nucleotides
• The base:
• can exist in 2 distinct orientations about the N-
glycosidic bond.
• These conformations :
• identified as, syn
• and anti.
• ‘Anti’ conformation predominates in naturally
occurring nucleotides.
Syn and anti conformations
syn-Adenosine anti-Adenosine
Nucleotides
• found in the cell primarily in their
phosphorylated form.
• The most common site of phosphorylation of
nucleotides found in cells:
• the hydroxyl group attached to the 5'-carbon of
the ribose.
Nucleotides
• The carbon atoms of the ribose present in
nucleotides:
• designated with a prime (') mark to distinguish
them from the backbone numbering in the
bases.
• Nucleotides can exist in the mono-, di-, or tri-
phosphorylated forms.
Nucleotides
• given distinct abbreviations to allow easy
identification of their structure,
• and state of phosphorylation.
• The monophosphorylated form of
adenosine (adenosine-5'-monophosphate):
• written as, AMP.
Nucleotides
• The di- and tri-phosphorylated forms:
written as, ADP and ATP, respectively.
• The use of these abbreviations assumes
that:
• the nucleotide is in the 5'-phosphorylated
form.
Nucleotides
• The di- and tri-phosphates of nucleotides:
linked by acid anhydride bonds.
• Acid anhydride bonds:
• have a high potential to transfer the
phosphates to other molecules.
• due to this property: the nucleotides that
results in their involvement in group
transfer reactions in the cell.
Nucleotides
• The nucleotides found in DNA:
• unique from those of RNA in that;
• the ribose exists in the 2'-deoxy form and the
abbreviations of the nucleotides:
• contain a d- designation.
• The monophosphorylated form of adenosine
found in DNA (deoxyadenosine-5'-
monophosphate) is written as dAMP.
Nucleotides
• The nucleotide uridine:
• never found in DNA and thymine is almost
exclusively found in DNA.
• Thymine: found in tRNAs but not rRNAs
nor mRNAs.
• There are several less common bases found
in DNA and RNA.
Nucleotides
• The primary modified base in DNA is 5-
methylcytosine.
• A variety of modified bases appear in the
tRNAs.
• Many modified nucleotides:
• encountered outside of the context of DNA
and RNA;
• that serve important biological functions.
Adenosine derivatives
• The most common adenosine derivative is the cyclic
form,
• 3'-5'-cyclic adenosine monophosphate, cAMP.
• This compound is a very powerful second
messenger,
• involved in passing signal transduction events from
the cell surface to internal proteins,
• e.g. cAMP-dependent protein kinase, PKA
Adenosine derivatives
cyclic form of GMP (cGMP) : also found in cells
involved as a second messenger molecule.
In many cases its' role is to antagonize the effects of
cAMP.
Formation of cGMP occurs in response to receptor
mediated signals similar to those for activation of
adenylate cyclase.
However, in this case :
guanylate cyclase coupled to the receptor.
Adenosine derivatives
☻The most important cGMP coupled signal transduction
cascade:
☻ photoreception.
☻ in this case activation of rhodopsin (in the rods);
☻ or other opsins (in the cones);
☻ by the absorption of a photon of light;
☻through 11-cis-retinal covalently associated with rhodopsin
and opsins
☻ activates transducin,
☻ which in turn activates a cGMP specific phosphodiesterase:
that hydrolyzes cGMP to GMP.
Synthetic nucleotide analogues
• Many nucleotide analogues :
• chemically synthesized and used for their therapeutic
potential.
• nucleotide analogues : can be utilized to inhibit specific
enzymatic activities.
Synthetic nucleotide analogues
• A large family of analogues are used as anti-
tumor agents:
• because they interfere with the synthesis of
DNA
• and thereby preferentially kill rapidly dividing
cells such as tumor cells.
Synthetic nucleotide analogues
• Some of the nucleotide analogues commonly
used in chemotherapy:
• 6-mercaptopurine,
• 5-fluorouracil,
• 5-iodo-2'-deoxyuridine
• 6-thioguanine.
Synthetic nucleotide analogues
• Each of these compounds :
• disrupts the normal replication process,
• by interfering with the formation of correct
Watson-Crick base-pairing.
Synthetic nucleotide analogues
• Nucleotide analogues also have been targeted for
use as antiviral agents.
• Several analogs are used to interfere with the
replication of HIV:
• such as AZT (azidothymidine),
• and ddI (dideoxyinosine).
Synthetic nucleotide analogues
• Several purine analogs: used to treat gout;
• e.g. most common : allopurinol;
• which resembles hypoxanthine.
• Allopurinol inhibits the activity of xanthine oxidase,
• an enzyme involved in de novo purine biosynthesis.
Synthetic nucleotide analogues
• Additionally, several nucleotide analogues
are used:
• after organ transplantation
• in order to suppress the immune system,
• and reduce the likelihood of transplant
rejection by the host.
Polynucleotides
• formed by the condensation of two or more
nucleotides.
• condensation most commonly occurs
between the alcohol of a 5'-phosphate of
one nucleotide;
• and the 3'-hydroxyl of a second,
• with the elimination of H2O, forming a
phosphodiester bond.
Polynucleotides
• The formation of phosphodiester bonds in
DNA and RNA:
• exhibits directionality.
• The primary structure of DNA and RNA
(the linear arrangement of the nucleotides):
• proceeds in the 5' ----> 3' direction.
Polynucleotides
• The common representation of the primary
structure of DNA or RNA:
• write the nucleotide sequences from left to
right synonymous with the 5' -----> 3'
direction as shown:
• e.g. 5'-pGpApTpC-3'
Structure of DNA
• Utilizing X-ray diffraction data, obtained from
crystals of DNA,
• James Watson and Francis Crick proposed a model
for the structure of DNA.
Structure of DNA
• This model predicted that DNA would exist:
• as a helix of two complementary antiparallel
strands,
• wound around each other in a rightward direction,
• and stabilized by H-bonding between bases in
adjacent strands.
Structure of DNA
• In the Watson-Crick model,
• the bases are in the interior of the helix
aligned at a nearly 90 degree angle relative
to the axis of the helix.
• Purine bases form hydrogen bonds with
pyrimidines,
• in the crucial phenomenon of base pairing.
Structure of DNA
• Experimental determination has shown that,
in any given molecule of DNA,
• the concentration of adenine (A) is equal to
thymine (T)
Structure of DNA
• and the concentration of cytidine (C) is
equal to guanine (G).
• This means that A will only base-pair with
T;
• and C with G.
Structure of DNA
• According to this pattern, known as Watson-
Crick base-pairing:
• the base-pairs composed of G and C contain
three H-bonds,
• whereas those of A and T contain two H-
bonds.
• This makes G-C base pairs more stable than
A-T base-pairs.
A-T Base Pair
G-C Base Pair
Structure of DNA
• The antiparallel nature of the helix:
• stems from the orientation of the individual
strands.
• From any fixed position in the helix,
• one strand is oriented in the 5' ---> 3'
direction
• and the other in the 3' ---> 5' direction.
Structure of DNA
• On its exterior surface,
• the double helix of DNA contains two deep
grooves:
• between the ribose-phosphate chains.
• These two grooves are of unequal size;
• and termed the major and minor grooves.
Structure of DNA
• The difference in their size is:
• due to the asymmetry of the deoxyribose
rings;
• and the structurally distinct nature of the
upper surface of a base-pair;
• relative to the bottom surface.
Structure of DNA
• The double helix of DNA (A-form)
• has been shown to exist in several different
forms,
• depending upon sequence content and ionic
conditions of crystal preparation.
Structure of DNA
• The B-form of DNA:
• prevails under physiological conditions of
low ionic strength;
• and a high degree of hydration.
Structure of DNA
• Regions of the helix that are rich in pCpG
dinucleotides:
• can exist in a novel left-handed helical
conformation termed Z-DNA.
• This conformation results from a 180 degree
change in the orientation of the bases,
• relative to that of the more common A- and B-
DNA.
B-DNA Z-DNA
Thermal properties of DNA
• As cells divide,
• it is a necessity that the DNA be copied
(replicated),
• in such a way that each daughter cell:
• acquires the same amount of genetic material.
Thermal properties of DNA
• In order for this process to proceed:
• the two strands of the helix must first be
separated,
• in a process termed ‘denaturation’.
• This process can also be carried out in vitro.
Thermal properties of DNA
• If a solution of DNA :
• subjected to high temperature, the H-bonds
between bases become unstable;
• and the strands of the helix separate in a
process of thermal denaturation.
Thermal properties of DNA
• The base composition of DNA:
• varies widely from molecule to molecule
• and even within different regions of the
same molecule.
• Regions of the duplex that have
predominantly A-T base-pairs:
• will be less thermally stable than;
• those rich in G-C base pairs.
Thermal properties of DNA
• In the process of thermal denaturation,
• a point is reached at which 50% of the DNA
molecule exists as single strands.
• This point is the melting temperature (TM),
• and characteristic of the base composition
of that DNA molecule.
Thermal properties of DNA
– The TM depends upon several factors in
addition to the base composition.
– These include the chemical nature of the
solvent;
– and the identities and concentrations of ions in
the solution.
Thermal properties of DNA
• When thermally melted DNA: cooled,
• the complementary strands will again re-form the
correct base pairs,
• in a process termed annealing or hybridization.
• The rate of annealing:
• dependent upon the nucleotide sequence of the
two strands of DNA.
Analysis of DNA structure
• Chromatography:
• Several of the chromatographic techniques
available for the characterization of
proteins;
• can also be applied to the characterization
of DNA.
• The most commonly used technique:
• HPLC (high performance liquid
chromatography).
Analysis of DNA structure
• Affinity chromatography technique can be also
employed.
• One common affinity matrix: hydroxyapatite (a
form of calcium phosphate),
• which binds double-stranded DNA;
• with a higher affinity than single-stranded DNA.
Electrophoresis:
• This procedure:
• serve the same function with regard to DNA
molecules;
• as it does for the analysis of proteins.
• However, since DNA molecules have much
higher molecular weights than proteins,
• the molecular sieve used in electrophoresis
of DNA must be different as well.
Electrophoresis:
• The material of choice for DNA:
• agarose, a carbohydrate polymer purified
from a salt water algae.
• is a copolymer of mannose and galactose
that when melted and re-cooled:
• forms a gel with pore sizes;
• dependent upon the concentration of
agarose.
Electrophoresis:
• The phosphate backbone of DNA:
• highly negatively charged,
• therefore DNA will migrate in an electric
field.
• The size of DNA fragments:
• can then be determined by comparing their
migration in the gel to known size
standards.
Pulsed-field gel electrophoresis
• Extremely large molecules of DNA (in
excess of 106 base pairs):
• effectively separated in agarose gels using
pulsed-field gel electrophoresis (PFGE).
Pulsed-field gel electrophoresis
• This technique employs two or more electrodes,
• placed orthogonally with respect to the gel,
• that receive short alternating pulses of current.
• PFGE allows whole chromosomes:
• and large portions of chromosomes to be analyzed.
LITERATURE CITED
• Devlin,T.M. Textbook of Biochemistry with
Clinical Correlations,Fifth Edition,Wiley-Liss
Publications,New York, USA, 2002.
• Lehninger, A. Principles of Biochemistry, Second
edition, Worth Publishers Co., New York, USA,
1993.
• Matthews, C.K. and van Holde, K.E.,
Biochemistry, Second edition, Benjamin /
Cummings Publishing Company Inc., San
Francisco, 1996.
