Nucleotides and Nucleic Acids

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Outline

1. Some Basics
2. Nucleic Acid Structure
3. Nucleic Acid Chemistry
4. Other Functions of Nucleotides

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 Nucleotides
• “Energy rich” compounds
– Chemical signals
– Enzyme co-factors

• Nucleic Acids
– DNA and RNA
– Polymers of nucleotides

• 3 components
– Nitrogenous “base”
– Ribose (or deoxyribose)
– Phosphate
Structure of nucleotides
Major purine bases of nucleic acids
Major pyrimidine bases of nucleic acids
Conformations of ribose
Conformations of ribose
Deoxyribonucleotides of nucleic acids
Ribonucleotides of nucleic acids
Nucleotide and nucleic acid nomenclature
Some adenosine monophosphates
 Polymerise Nucleotides

• nucleotides can be linked

– phosphates linked to 2 pentoses
– phosphodiester linkages
• Link PO4 at
– 5’ end to 3’ OH of next nucleotide

• chain has POLARITY

–distinct ends
•5’ end
•3’ end
–usually “read” 5’ -> 3’
 Nucleotides as Energy Carriers
• ATP
– Adenosine triphosphate
• ADP, AMP
– Adenosine diphosphate
– Adenosine monophosphate
ATP  ADP + PO4
ADP  AMP + PO4
• Main energy exchange reactions in
cells
Hydrolysis of RNA under alkaline conditions
• Sugar, D-ribose or 2’-deoxy D-ribose, is linked
to base via a covalent β-N-glycosidic bond to
N-9 of a purine or to N-1 of a pyrimidine
• Nucleotides are termed ribonucleotides or
deoxyribonucleotides based on whether the
sugar is ribose or 2’-deoxyribose
Tautomeric forms of uracil
Schematic representation of nucleotide
sequence
Absorption spectra of common nucleotides
Nucleotides are phosphorolated nucleosides

• Mononucleotides are nucleotides singly

phosphorolated on hydroxyl group of the
sugar
• AMP → Adenosine monophosphate
• Adenine + ribose +phosphate
• Additional phosphates linked by acid
anhydride bonds to the existing phosphate of a
mononucleotide form nucleoside di- and
triphosphates
• ADP → Adenosine diphosphate
• Adenine + ribose +phosphate +phosphate

• ATP → Adenosine triphosphate

• Adenine + ribose +phosphate +phosphate +
phosphate
 Functions of nucleotides
• Nucleic acid biosynthesis
• Energy production and transduction
• Protein biosynthesis
• Regulatory cascades
• İntra- and intercellular signal transduction
• Biosynthesis some biomolecules
 Some properties of nucleotides

• [Link] have a negative charge at

physiological pH
• [Link] absorb UV light
• 3. Many coenzymes are nucleotide derivatives
• 4. Synthetic nucleotide analogs are used in
chemotherapy
• 5. Nucleoside triphosphates have high group transfer
potential
• [Link] nucleotides are involved in signal transduction
 Polynucleotides
• Mononucleotides are covalently linked through phosphate-group
‘bridges’
• Specifically, the 5’-OH group of one nucleotide unit is joined to
the 3’-OH group of the next nucleotide by a phosphodiester
linkage resulting to release of one molecule water
• This results in a dinucleotide
• The term oligonucleotide is used for polymers containing 50 or
fewer nucleotides
• Polynucleotides are directional macromolecules
• Since the phosphodiester bond links 3’- and 5’- carbons of
adjacent monomers, each end of a polimers is distinct. They are
reffered as ‘3’-end’ or ‘5’-end’ of polynucleotides
• In the most representations displaying only the base sequences 5’
end is shown on the left and 3’ end is shown on the right:
5’ GTATTGC 3’
• Nucleic acids are the polimers containing many nucleotides linked
by phosphodiester linkage
• Hydrolysis of polynucleotides has a large energy barrier and this
reaction is too slow in the absence of phosphodiesterases
• Phosphodiester linkes are hydrolyzed by phosphodiesterases

•
 Nucleic acid structure and function
• Genetic information is coded by DNA
• Genes control the synthesis of various types of RNA which are
involved in protein synthesis
• Nucleic acids (DNA and RNA) are the polimers containing
nucleotides as monomers.
• Covalent backbones of nucleic acids consist of alternating
phosphate and pentose residues, and the characteristic bases
may be regarded as side groups joint to the backbone at regular
intervals
• Nucleic acids contain five major heterocyclic base:
• Adenine (A), guanine (G), cytosine (C), tymine (T) and uracil (U)
• DNA includes A,G,C,T
• RNA includes A,G,C,U
 Structure of DNA?
• The Genetic Material
• Crick and Watson
– Race with Linus Pauling to predict structure

• Chargaff’s rules:
– Chemical analysis:
[A] = [T]
[G] = [C]
– Constant
• for each organism
– over time
– across all tissues
The Avery-Macleod-McCarty experiment
The Hershey-Chase experiment
 X-Ray Diffraction
• Predict
– Double helix
– 2 periodicities
• 3.4Å
• 34Å
Hydrogen-bonding patterns in the base pairs
defined by Watson and Crick
• This common form of DNA is said to be right- handed because as
one looks down the double helix the base residues form a spiral in a
clockwise direction
• The predominant tautomers of the four bases allow A to pair only
with T, and G only with C
• The two strands of the double-helical molecule are antiparallel; one
strand runs in the 3’-5’ direction while the other in the 5’-3’
direction
• In the double-stranded DNA molecules, genetic information resides
in the sequence of nucleotides on one strand, template strand
• This is the strand of DNA that is copied during nucleic acid synthesis
(noncoding strand)
• The opposite strand is considered the coding strand because it
matches the RNA transcript that encodes the protein
•
 The Double Helix
• 3.4Å per basepair
• 10 basepairs per turn
– 10-11 in aqueous solution
• 2 anti-parallel strands
• The two strands wind around a central axis in the form of a
double helix
• Double-stranded DNA exists in at least six forms (A-E and Z)
• The B form is usually found under physiologic conditions
• A single turn of B-form DNA about the axis of the molecule
contains ten base pairs
• The distance spanned by one turn of B-form DNA is 3.4 nm, the
helical diameter of the double helix is 2 nm
• Three hydrogen bonds hold the deoxyguanosine nucleotide to
the deoxycytidine nucleotide, whereas A and T are helded by
two hydrogen bonds
• G-C bonds are much more resistant to denaturation than A-T
bonds
•
Watson-Crick model for the structure of DNA
Complementarity of strands in the DNA
double helix
Replication of DNA as suggested by
Watson and Crick
Structural variation in DNA
Comparison of A, B, and Z forms of DNA
Comparison of A, B, and Z forms of DNA
Palindromes and mirror repeats
Hairpins and cruciforms
DNA structures containing three of four
DNA strands- Hoogsteen pairing

Hoogsteen pairing
DNA structures containing three of four
DNA strands- Guanosine tetraplex
DNA structures containing three of four
DNA strands- H-DNA
 The denaturation of DNA (melting)
• Double stranded DNA can be separated into two component strands
in solution by increasing the temperature or decreasing the salt
concentration
• As a result of denaturation, optical absorbance of bases increases
• Double stranded DNA molecule exhibits properties of a rigid rod and
in solution is a viscous material because of the stacking of the bases
and hydrogen bonding but it loses its viscosity upon denaturation
• DNA rich in G-C pairs melts at a higher temperature than that rich in
A-T pairs
• Separated strands of DNA will renature when physiologic
temperature and salt conditions are achieved
• The rate of reassociation depends upon the concentration of the
complementary strands
• There are grooves in the DNA molecule
• A major groove and a minor groove wind along the molecule
parallel to the phosphodiester backbones.
• In these grooves, regulatory proteins can interact specifically with
exposed atoms of the nucleotides
• Therefore these proteins recognize and bind to specific nucleotide
sequences without disturbing the base pairing
• Regulatory proteins can control the expression of specific genes via
such interactions
• DNA exists in relaxed and supercoiled forms
• In bacteria and many DNA containing animal viruses the ends of the
DNA molecules are joined to create a closed circle with no terminal
•
This structure does not destroy polarity but eliminates all free 3’
and 5’ hydroxyl and phosphoryl groups
•Closed circles may exist in relaxed or supercoiled form
•Supercoils are introduced when a closed circle is twisted
around its own axis or when a linear piece of dublex DNA is
twisted
•This is an energy-requiring process and puts the DNA under
stress
•Negative supercoils are formed when the molecule is twisted in
the opposide direction of clockwise turns of the right-handed
double helix found in B-DNA. Such DNA is said to be
underwound
• The transition to another form is required energy
• One such transition occurs during the strand separation just
before the replication
• Enzymes that catalyze topologic changes of DNA are called as
topoisomerases
• Topoisomerases can relax or insert supercoils using ATP
• The best characterized is bacterial gyrase which induced
negative supercoilig in DNA
Reversible denaturation and annealing
(renaturation) of DNA
Heat denaturation of DNA
Partially denatured DNA
 Chemical structure of RNA
• The chemical nature of RNA differs from that of DNA
• RNA is also formed by purines and pyrimidines linked by 3’-5’
phosphodiester bonds
• Although sharing many features with DNA , RNA possesses several
specific differences:
• 1. In RNA, the sugar moiety to which the phosphates and purine and
pyrimidines are attached is ribose instead of deoxyribose of DNA
• 2. The pyrimidine components of RNA is differ from those of DNA. RNA
contains the A, G,C but does not contain T (with a rare exception),
instead of T, U is present in RNA
• [Link] exist as a single strand. However, given the proper
complementary base sequence with opposite polarity, single strand RNA
is capable of folding back on itself like a hairpin thus acquiring double-
stranded structure
Three-dimensional structure in RNA
Prokaryotic mRNA
Typical right-handed stacking pattern of
single-stranded RNA
 RNA
• Usually single stranded
• Genetic material of RNA virus
• Functional:
– e.g. Translation machinery
• rRNA (ribosomal RNA)
• tRNA (transfer RNA)
• Regulatory:
– Control of gene expression
• miRNA (microRNA)
• Gene Expression
– mRNA (messenger RNA)
– Copy of 1 gene for translation by ribosomes
Secondary structure of RNAs
 RNA Secondary Structure
• Many functional RNAs
have secondary structure
• G-U basepairs allowed
• 4. Since the RNA molecule is a single strand complementary to only one of the
two strands of a gene, its guanine content does not necessarily eaqual its
cytosine content; and its adenine content does not necessarily eaqual its uracil
content
• 5. RNA can be hydrolyzed by alkali to 2’, 3’cyclic diesters of mononucleotides
but those molecules can not be formed from alkali-treated DNA because of
the absence of a 2’- OH group
• The alkali lability of RNA is useful both diagnostically and analytically
• Information within the RNA is contained in its sequence of purine and
pyrimidine nucleotides.
• The sequence is complementary to the template strand of the gene that was
transcribed
• Because of this complementarity, a RNA molecule can bind specifically via the
base-pairing rules to its template DNA strand; but it does not bind to coding
strand
• The nucleotide sequence of the RNA molecule is the same as that of the
coding strand of the gene, except for U replacing T
 RNA types and their funtions
• Cytoplasmic RNA molecules that serve as a templates for protein
synthesis are designated as messnger RNAs (mRNA). mRNA
molecules transfer genetic information from DNA to protein-
synthesizing machinary
• Many other cytoplasmic RNA molecules have structural roles.
They contribute to the formation of ribosomes (ribosomal RNA,
rRNA) or serve as adapter molecules (transfer RNA, tRNA) for the
translation of RNA information into specific sequences of
polymerized amino acids
• Some RNA molecules have intrinsic catalytic activity. The activity
of these ribozymes often involved in the cleavage of a nucleic
acid
• In human cells there are small nuclear RNA (snRNA) species.
These are not directly involved in protein synthesis but that
may have roles in RNA processing
• These relatively small molecules vary in size from 90 to 300
nucleotides
• The genetic material for some animal and plant viruses is RNA
rather than DNA
• Many animal RNA viruses are, retroviruses, transcribed by an
RNA-dependent DNA polymerase , the so-called reverse
transcriptase, to produce a double- stranded DNA copy of
their RNA genome
• In many cases, the resulting double- stranded DNA transcript
is integrated into the host genome and subsequently serves
as a template for gene expression and from which new viral
RNA genomes can be transcribed
•
• A newly synthesized RNA molecule is called as primary
transcript. Primary transcript is processed depending on
the type of RNA
• In all prokaryotic and eukaryotic organisms, three main
classes of RNA molecules exist. Each differs from the
others by size, function and general stability
 1. Messenger RNA (mRNA)
• This is the most heterogenous class in size and stability
• All members of the class function as messengers conveying
the information in a gene to the protein-synthesizing
machinery
• Each serves as a template for a specific sequence of amino
acids that is polimerized to form a specific protein
• mRNAs have some unique chemical characteristics:

• The 5’ terminal of mRNA is capped by a 7-methylguanosine

triphosphate
• The cap is involved in the recognition of mRNA by translating
machinery, and it probably helps stabilize the mRNA by preventing
the attack of 5’-exonucleases
• The protein synthesis begins translating the mRNA into proteins at
the 5’or capped terminal
• The other end of most mRNAs has attached a polymer of
adenylate residues 20-250 nucleotides in lenght so called the poly
A tail
• The funtion of the poly A tail seems that it maintains the stability
of the mRNA by preventing the attack of 3’-exonucleases
• Polynucleotides are directional macromolecules
• Since the phosphodiester bond links 3’- and 5’- carbons of
adjacent monomers, each end of a polimers is distinct. They are
reffered as ‘3’-end’ or ‘5’-end’ of polynucleotides
• In the most representations displaying only the base sequences 5’
end is shown on the left and 3’ end is shown on the right:
• 5’ GTATTGC 3’
•
 2. Transfer RNA (tRNA)
• tRNA molecules vary in lenght from 74 to 95 nucleotides
• tRNA molecules serve as a adapters for the translation of the
information in the sequence of nucleotides of mRNA into specific
amino acids
• There are at least 20 species of tRNA molecules in every cell. At
least one, often several corresponding to each of the 20 amino
acid are required for protein synthesis
• Although each specific tRNA differs from the others in its sequence
of nucleotides, the tRNA molecules as a class have many features
in common
• The primary structure of all tRNA molecules allows extensive
folding and intrastrand complementarity to generate a secondary
structure
• This structure appears like a clover leaf
• All tRNA molecules contain four main arms
• The acceptor arm consists of a base-paired stem that terminates
in the sequence CCA (5’ to 3’). It is through an ester bond to the 3’
hydroxyl group of the adenosyl moiety that the carboxyl groups of
amino acids are attached
• The other arms have base-paired stems and unpaired loops
• The anticodon arm at the end of a base-paired stem recognizes
the triplet nucleotide or codon of the template mRNA. It has a
nucleotide sequence complementary to the codon and is
responsible for the specifity of the tRNA
• The D arm is named for the presence of the base dihydrouridine,
and the TΨC arm for the sequence T, pseudouridine, and C
•
• The extra arm is the most variable feature of tRNA. It accounts for
the differences in lenght of the tRNAs; and it provides a basis for
classification.

• Class 1 tRNAs have an extra arm that is 3-5 bp long

• Class 2 tRNAs have an extra arm that is 13-21 bp long
• The secondary structure of tRNA molecules is maintained by the
base pairing in these arms and this is a consistent feature :
• The TΨC and anticodon arms have 5 bp
• The D arm has 3-4 bp
• The acceptor arm has 7 bp
•
 3. Ribosomal RNA (rRNA)

• A ribosome is a cytoplasmic nucleoprotein structure that acts as

the machinery for the synthesis of proteins from the mRNA
templates
• On the ribosomes, the mRNA and tRNA molecules interact to
translate into a specific protein molecule information
transcribed from the gene
• During the active protein synthesis, many ribosomes are
associated with an mRNA molecule in an assembly called the
polysome
• The mammalian ribosome contains two major nucleoprotein
subunits, a larger one with a molecular weight of 2.8x106
(sedimentation velocity is 60S*) and a smaller subunit with a
molecular weight of 1.4x106 (40S)
•
• The 60S subunit contains a 5S ribosomal RNA, a 5.8S rRNA and a28S
rRNA; there are also probably more than 50 specific polypeptides
• *Svedberg unit (sedimentation coefficient)
• The 40S subunit is smaller and contains 18S rRNA and approximately
30 polypeptide chains
• All of the rRNA molecules, except the 5S rRNA are processed from a
single 45S precursor RNA molecule in the nucleus
• 5S rRNA has its own precursor that is independently transcribed
• The highly methylated rRNA molecules are packaged in the nucleus
with the specific ribosomal proteins; but in the cytoplasm, ribosomes
remain quite stable and capable of many translaton cycles
• Ribosomal RNA molecules are necessary for ribosomal assembly and
seem to play key roles in the binding of mRNA to ribosomes and its
translation