Chap.

8A Nucleotides and Nucleic Acids

• Some Basics
• Nucleic Acid Structure
• Nucleic Acid Chemistry
• Other Functions of Nucleotides

Fig. 8-12. X-ray diffraction pattern of DNA

fibers.
 Intro. to Nucleotides and Nucleic Acids
Nucleotides have a variety of roles in cellular metabolism. They are
the energy currency in metabolic reactions, the essential chemical
links in the response of cells to hormones and other stimuli, and the
structural components of a variety of enzyme cofactors and
metabolic intermediates. They are also constituents of the nucleic
acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The
amino acid sequence of every protein in a cell, and the nucleotide
sequence of every RNA, is specified by a nucleotide sequence in
genomic DNA. Segments of DNA specifying the synthesis of a
functional protein or RNA product are called genes. The storage and
transmission of biological information are the only known functions
of DNA. RNAs have a broader range of functions. Ribosomal RNAs
(rRNAs) are structural and catalytic components of ribosomes.
Messenger RNA (mRNAs) carry genetic information specifying the
sequences of proteins. Transfer RNAs (tRNAs) are adaptor
molecules that participate in translating the information in mRNA
into a specific sequence of amino acids in a polypeptide. There is
also a wide variety of special-function RNAs, including some called
ribozymes that have enzymatic activity.
 Structure of Nucleotides
Nucleotides contain three components: 1) a nitrogen-containing
base, 2) a pentose, and 3) one or more phosphates (Fig. 8-1a). In
the absence of the phosphate group(s), the molecule is called a
nucleoside. The nitrogenous bases are derivatives of pyrimidine and
purine. The numbering of the ring atoms of pyrimidines and purines
is illustrated in Fig. 8-1b. The numbering of the pentose rings
follows the convention outlined in Chap. 7, except that the carbon
numbers in the pentoses of nucleotides and nucleosides are given a
prime (‘) designation to distinguish them from the numbered atoms
of the bases. The base of a nucleotide is joined covalently (at N-1
of pyrimidines and N-9 of purines) in an N-ß-glycosyl bond to the
1’ carbon of the pentose, and the phosphate is esterified commonly
to the 5’ carbon. Water is removed in the formation of the N-ß-
glycosyl bond as occurs in O-glycosidic bond formation.
 Major Bases of Nucleic Acids
Both DNA and RNA contain two major purine bases, adenine (A)
and guanine (G) (Fig. 8-2). Both nucleic acids also contain the
pyrimidine, cytosine (C), and a second pyrimidine that is thymine
(T) in DNA and uracil (U) in RNA. Only occasionally does thymine
occur in RNA or uracil in DNA. In some cases the names of the
bases reflect the sources from which they originally were isolated.
Guanine, for example, was first isolated from guano (bird manure)
and thymine was first isolated from thymus tissue.
Nomenclature of Nucleosides & Nucleotides
The names of the nucleosides and nucleotides containing the five
common bases are listed in Table 8-1.
 The Pentoses of Nucleotides
Nucleotides have two kinds of pentoses. The recurring
deoxyribonucleotide units of DNA contain 2’-deoxy-D-ribose, and
the ribonucleotide units of RNA contain D-ribose. In both types
of nucleotides the pentoses exist in their ß-furanose (closed five-
membered ring) forms. The formation of the ß-D-ribofuranose
ring from the straight-chain aldehyde form of D-ribose in solution
is illustrated in Fig. 8-3a. Deoxyribose undergoes a similar
interconverion in solution, but in DNA exists solely as ß-2’-deoxy-
D-ribofuranose. Deoxyribofuranose and ribofuranose rings in
nucleotides exist in four different puckered conformations (Fig. 8-
3b). In all cases, four of the five ring atoms are nearly in the
same plane. The fifth atom (C-2’ or C-3’) is on either the same
(endo) or the opposite (exo) side of the plane relative to the C-5’
atom.
 Deoxyribonucleotides of DNA
The structures and names of the four major deoxyribonucleotides
(deoxyribonucleoside 5’-monophosphates) of DNA are shown below
(Fig. 8-4a). All nucleotides are shown in their free form at pH
7.0. The deoxyribonucleotide units of DNA are usually symbolized
as A, G, T, and C, and sometimes as dA, dG, dT, and dC. In their
free forms, the deoxyribonucleotides are commonly abbreviated
dAMP, dGMP, cTMP, and dCMP. For each nucleotide in the figure,
the more common name is followed by the complete name in
parentheses. All abbreviations assume that the phosphate group is
at the 5’ position. The nucleoside portion of each molecule is
shaded in light red.
 Ribonucleotides of RNA
The structures and names of the four major ribonucleotides
(ribonucleoside 5’-monophosphates) of RNA are shown below (Fig.
8-4b). All nucleotides are shown in their free form at pH 7.0.
The ribonucleotide units of RNA are usually symbolized as A, G,
U, and C. In their free forms, the ribonucleotides are commonly
abbreviated AMP, GMP, UMP, and CMP. For each nucleotide in the
figure, the more common name is followed by the complete name
in parentheses. All abbreviations assume that the phosphate group
is at the 5’ position. The nucleoside portion of each molecule is
shaded in light red.
Minor Bases
Both DNA and RNA also contain some
minor bases (Fig. 8-5). In DNA, the
most common of these are methylated
forms of the major bases (Panel a).
Minor bases of many types occur in
tRNAs (Panel b). If the modification
occurs directly on one of the ring
atoms of the pyrimidine or purine base,
the convention is to simply indicate the
ring position of the substituent by its
number, e.g., 5-methylcytidine. The
convention changes when the
substituent atom is exocyclic (not within
the ring structure). In this case the
type of atom is identified and the ring
position to which it is attached is
denoted with a superscript, e.g., N6-
methyladenosine.
 Some Adenosine Monophosphates
Cells also contain nucleotides with phosphate groups in positions
other than on the 5’ carbon of the pentose ring (Fig. 8-6). For
example, ribonucleoside 2’,3’-cyclic monophosphates are isolatable
intermediates, and ribonucleoside 3’ & 2’-monophosphates are end
products of the hydrolysis of RNA by certain ribonucleases. Other
variants are adenosine 3’,5’-cyclic monophosphate (cAMP), which is
a very important molecule in some signal transduction pathways.
 Phosphodiester Linkages in the Covalent
Backbone of DNA and RNA
The successive nucleotides in DNA and
RNA are covalently linked through
phosphate-group bridges in which the 5’-
phosphate of one nucleotide unit is joined
to the 3’-hydroxyl group of the next,
creating a phosphodiester linkage (Fig. 8-
7). Thus, the covalent backbones of
nucleic acids consist of alternating
phosphate and pentose residues, and the
nitrogenous bases may be regarded as side
groups joined to the backbone at regular
intervals. The backbones of both DNA and
RNA are hydrophilic. The hydroxyl groups
of the sugar residues form hydrogen bonds
with water. The phosphate groups, with a
pKa near 0, are completely ionized and
negatively charged at pH 7. The negative
charges are generally neutralized by ionic
interactions with positive charges on
proteins, metal ions, and polyamines. All
the phosphodiester linkages in DNA and
RNA have the same orientation along the
chain giving each linear nucleic acid strand
a specific polarity and distinct 5’ and 3’
ends. By definition, the 5’ end lacks a
nucleotide at the 5’ position and the 3’ end
lacks a nucleotide at the 3’ position.
 Hydrolysis of RNA by Alkali
The covalent backbone of DNA and RNA is subject to slow,
nonenzymatic hydrolysis of its phosphodiester bonds. In vitro, RNA
is hydrolyzed rapidly under alkaline conditions, but DNA is not.
This is because the 2’-hydroxyl group in the ribose moieties of
RNA is directly involved in the cleavage process (Fig. 8-8). 2’,3’-
cyclic monophosphate nucleotides are the first products of the
action of alkali on RNA and are subsequently hydrolyzed further to
yield a mixture of 2’- and 3’-nucleoside monophosphates.
 Representations of Nucleotides
The nucleotide sequences of nucleic acids can be represented as
illustrated below for a segment of DNA with five nucleotide units.
The phosphate groups are symbolized by circled Ps, and each
deoxyribose is symbolized by a vertical line, from C-1’ at the top
to C-5’ at the bottom. The connecting lines between nucleotides
(which pass through the P symbols) are drawn diagonally from the
middle (C-3’) of the deoxyribose of one nucleotide to the bottom
(C-5’) of the next. Some simpler representations of this
pentadeoxyribonucleotide are pA-C-G-T-AOH, pApCpGpTpA, and
pACGTA. Note that the sequence of a single strand of nucleic acid
is always written with the 5’ end at the left and the 3’ end at the
right, that is in the 5’3’ direction. A short nucleic acid such as
shown in the figure is referred to as an oligonucleotide. This term
is generally applied to nucleotides of 50 or fewer residues. Longer
nucleic acids are referred to as polynucleotides.
 Tautomeric Forms of Uracil
Free pyrimidine and purine bases may exist in two or more
tautomeric forms depending on the pH. Uracil, for example,
occurs in lactam, lactim, and double lactim forms depending on
the pH (Fig. 8-9). Certain tautomeric forms predominate at
neutral pH, and these are the structures shown for the five
common purines and pyrimidines in Fig. 8-2. These are the
tautomers that are present in the bases in DNA and RNA.
 Nucleotide Absorption Spectra
All nucleotide bases absorb UV light, and nucleic acids are
characterized by a strong absorption at wavelengths near 260 nm
(Fig. 8-10). Plotted in this figure is the variation in molar
extinction coefficient, , as a function of wavelength. The molar
extinction coefficients at 260 nm are listed in the attached table.
The spectra of corresponding ribonucleotides and
deoxyribonucleotides are essentially identical. For mixtures of
nucleotides, a wavelength of 260 nm is used for absorption
measurements.
 Watson and Crick Base-pairing in DNA
The functional groups of pyrimidines and purines are ring nitrogens,
carbonyl groups, and exocyclic amino groups. Hydrogen bonds
involving the amino and carbonyl groups are the most important
mode of interactions between two complementary strands of nucleic
acid. The most common hydrogen-bonding patterns are those
defined by Watson and Crick in 1953, in which A bonds specifically
to T (or U) and G bonds to C (Fig. 8-11). These two types of base
pairs predominate in double-stranded DNA and RNA, and the
tautomers shown in Fig. 8-2 are responsible for these types of
base pairs. It is this specific pairing of bases that permits the
duplication of genetic information in DNA, as discussed below.
Watson-Crick Model for the Structure of
Double-helical DNA (I)
A model for the structure of DNA was proposed by Watson and
Crick in 1953. Their model was based on a number of pieces of
information that were available at the time about the composition
of DNA and the x-ray diffraction properties of DNA fibers. Most
importantly, x-ray diffraction studies of DNA fibers performed
by Rosalind Franklin and Maurice Wilkins (Fig. 8-12) showed that
DNA molecules are helical and exhibit two periodicities repeating
along the length of the fiber--a primary repeat of 3.4 Å and a
secondary repeat of 34 Å. In addition, Erwin Chargaff and
colleagues had shown through DNA compositional analysis that the
number of T residues always equals the number of A residues (A =
T), and the number of G residues always equals the number of C
residues (G = C). As a result, the sum of purine residues equals
the sum of pyrimidine residues (A + G =
T + C). Watson and Crick then set
about to develop a structure that was
consistent with these two sets of data
(next slide).
 Watson-Crick Model for the Structure of
Double-helical DNA (II)
In DNA, Watson and Crick proposed
that two helical DNA chains are wound
around the same axis to form a right-
handed double helix (Fig. 8-13). They
speculated that the two chains have an
antiparallel orientation, and this was
later proven to be true. The
hydrophilic backbones of alternating
deoxyribose and phosphate groups are
on the outside of the helix facing the
surrounding water. The furanose ring
of each deoxyribose is in the C-2’
endo conformation. The purine and
pyrimidine bases of both strands are
stacked inside the double helix with
their hydrophobic and nearly planar
ring structures very close together and
perpendicular to the axis of the helix.
(Continued on the next slide).
 Watson-Crick Model for the Structure of
Double-helical DNA (III)
Base stacking accounts for the 3.4 Å
repeat along the length of the helix.
The secondary repeat of about 34 Å
was accounted for by the presence of
10 base pairs in each complete turn of
the double helix. This was later
modified to 10.5 base pairs per turn
for DNA in aqueous solution. In the
Watson-Crick model, A/T and G/C base
pairing was proposed based on the fact
that these combinations of bases fit
well inside the double helix. Finally, the
offset pairing of the two strands
creates a major and a minor groove on
the surface of the duplex. It should be
noted that the double helix not only is
stabilized by Watson-Crick base pairing
between residues in the helix, but is
also stabilized by base-stacking
interactions that remove the bases from
contact with water. The features of the
double-helical model of DNA structure
are supported by much chemical and
biochemical evidence.
 Double-helical Strand Complementarity
The two antiparallel chains of double-helical
DNA are not identical in either base sequence
or composition. Instead, they are
complementary to one another. Wherever
adenine occurs in one chain, thymine occurs in
the other. Similarly, guanine occurs opposite
cytosine in the two chains (Fig.8-14).
Watson-Crick Model for DNA Replication
The model for DNA structure
immediately suggested to Watson and
Crick a mechanism for the transmission
of genetic information. The essential
feature of the model is the
complementarity of the two DNA strands
in the double helix. As Watson and Crick
were able to see, well before
confirmatory data became available, this
structure could logically be replicated by
separating the two strands, and
synthesizing a complementary strand for
each (Fig. 8-15). Because nucleotides in
each strand are joined in a sequence
specified by the base-pairing rules
stated above, each preexisting strand
functions as a template to guide the
synthesis of one complementary strand.
Worked Example 8-1. Base Pairing in DNA
 Structural Variation in DNA
Double-helical DNA is a very flexible molecule. Considerable
rotation is possible around several types of bonds in the
phosphodeoxyribose backbone, and thermal fluctuation can produce
bending, stretching, and unpairing (melting) of the strands. Many
significant deviations from the Watson-Crick DNA structure are
found in cellular DNA, some or all of which may be important in
DNA metabolism. Note that these structural variations generally do
not affect the fundamental properties of DNA, such as strand
complementarity, antiparallel strands, and the requirement for A/T
and G/C base pairs. Structural variations in DNA are due to three
things: the different possible conformations of the deoxyribose,
rotation about the contiguous bonds that make up the
phosphodeoxyribose backbone (Fig. 8-16a), and free rotation about
the C-1’-N-glycosyl bond (Fig. 8-16b). Because of steric
constraints, purines in purine nucleotides are restricted to two
stable conformations with respect to deoxyribose, called syn and
anti (Fig. 8-16b). Pyrimidines generally are restricted to the anti
conformation because of steric interference between the sugar and
the carbonyl oxygen at C-2 of the pyrimidine.
 The A, B, and Z Forms of DNA (I)
The Watson-Crick structure of DNA
is also referred to as B-form DNA,
or B-DNA. B-DNA is the most stable
structure for a random-sequence DNA
molecule under physiological conditions
and is therefore the standard
structural reference in any study of
the properties of DNA. Two structural
variants that have been well
characterized in crystal structures
are the A- and Z-forms of DNA (Fig.
8-17). The properties of these forms
are summarized in the Fig. 8-17 table
(next slide). In general, A-DNA is a
dehydrated form of DNA that may
not occur in cells. A similar type of
structure does occur in double helical
RNA. There is evidence for some
short tracts of Z-DNA in bacterial
and eukaryotic cells. These Z-DNA
tracts may play a role (as yet
unidentified) in regulating the
expression of some genes or in genetic
recombination.
 The A, B, and Z Forms of DNA (II)
The DNA backbone of Z-DNA takes on a zigzag conformation.
Certain nucleotide sequences fold into left-handed Z helices much
more readily than others. Prominent examples are sequences in
which pyrimidines alternate with purines, especially alternating C
and G or 5-methyl-C and G residues. To form the left-handed
helix in Z-DNA, the purine residues flip to the syn conformation,
alternating with pyrimidines in the anti conformation.
DNA Palindromes and Mirror Repeats (I)
It is thought that other sequence-dependent structural variations
found in larger chromosomes may affect the function and
metabolism of the DNA segments in their immediate vicinity. A
rather common type of such DNA sequence is a palindrome. A
palindrome is a word, phrase, or sentence that is spelled
identically read either forward or backward. A example is the
word ROTATOR. The term is applied to regions of DNA with
inverted repeats of base sequence having twofold symmetry over
two strands of DNA (Fig. 8-18). As shown in the next slide, such
sequences have the potential to form unique DNA structures. When
the inverted repeat occurs within each individual strand of the
DNA, the sequence is called a mirror repeat. Mirror repeats
cannot form the same types of structures as DNA palindromes.
DNA Palindromes and Mirror Repeats (II)
DNA palindromes are self-complementary
within each strand and therefore have the
potential to form hairpin and cruciform
(cross-shaped ) structures (Fig. 8-19).
Sequences of these types are found in
virtually every large DNA molecule and can
encompass a few base pairs or thousands.
The extent to which palindromes occur in
cruciforms in cells is not known, although
some cruciform structures have been
demonstrated in vivo in E. coli. Self-
complementary sequences cause isolated
single strands of DNA (or RNA) in solution
to fold into complex structures containing
multiple hairpins.
 Triple-helical DNA (I)
Several unusual DNA structures involve three or four DNA strands.
Nucleotides participating in a Watson-Crick base pair (Fig. 8-11)
can form additional hydrogen bonds, particularly with functional
groups in the major groove. For example, a cytidine residue if
protonated can pair with a guanosine residue of a G/C base pair,
and an thymidine can pair with the adenosine of an A/T pair (Fig.
8-20a). The N-7, O6, and N6 of purines are the atoms that
participate in the hydrogen bonding of triplex DNA. These atoms
are often referred to as Hoogsteen positions, and the non-Watson-
Crick pairing is called Hoogsteen pairing after their discoverer,
Karst Hoogsteen. This type of base pairing allows the formation of
triplex DNAs.
 Triple-helical DNA (II)
Some triplex DNAs contain two pyrimidine strands and one purine
strand. Others contain two purine strands and one pyrimidine
strand. In Fig. 8-20b, triple-helical DNA containing two pyrimidine
strands (red and white; sequence TTCCT) and one purine strand
(blue; sequence AAGGAA) are shown. The blue and white strands
are antiparallel and are paired by normal Watson-Crick base pairs.
The third (all pyrimidine strand) is parallel to the purine strand and
paired through non-Watson-Crick hydrogen bonds. The triplexes
shown in Fig. 8-20a&b are most stable at low pH because the
C/G.C+ triplet requires a protonated cytosine. In the triplex, the
pKa of this cytosine is >7.5, which is raised considerably from its
normal value of 4.2. Triplexes form most readily within long
sequences containing only pyrimidines or only purines in a given
strand.
 Tetraplex DNA
Four DNA strands can also pair to form a tetraplex (quadruplex),
but this occurs readily only for DNA sequences with a very high
proportion of guanosine residues (Fig. 8-20c&d). The guanosine
tetraplex, or G tetraplex, is quite stable over a wide range of
conditions. The orientation of strands in the tetraplex can vary
as shown in Fig. 8-20e.
 Intro. to RNA Structure
As in the case of protein structure, it is sometimes useful to
describe nucleic acid structure in terms of hierarchical levels of
complexity (i.e., primary, secondary, and tertiary structure). The
primary structure of a nucleic acid is its covalent structure and
nucleotide sequence. Any regular, stable structure taken up by
some or all of the nucleotides in a nucleic acid can be referred to
as secondary structure. The complex folding of large chromosomes
within eukaryotic chromatin and the bacterial nucleoid, or the
elaborate folding of large tRNA or rRNA molecules, is generally
considered tertiary structure. The process by which RNAs are
formed on a DNA template is known as transcription. In bacteria,
mRNAs can contain the protein coding sequences (red) for one gene
(monocistronic) or multiple genes (polycistronic) (Fig. 8-21). mRNAs
also contain noncoding regions (gray) at their ends and between
protein coding sequences that may be involved in regulation of
protein synthesis.
 Base Stacking in Single-stranded RNA
The product of transcription of DNA is
always single-stranded RNA. The single
strand tends to assume a right-handed
helical conformation dominated by base-
stacking interactions (Fig. 8-22). In this
structure the bases are shown in yellow, the
phosphorous atoms in orange, and the ribose
and phosphate oxygens in green. Base
stacking interactions are stronger between
two purines than between a purine and
pyrimidine or between two pyrimidines. The
purine-purine interaction is so strong that a
pyrimidine separating two purines is often
displaced from the stacking pattern so that
the purines can interact.
 RNA Secondary Structures (I)
Any self-complementary sequences in single-stranded RNAs produce
more complex secondary structures (Fig. 8-23). Base pairing in RNA
matches the pattern observed for DNA. Namely, G pairs with C,
and A pairs with U (or the occasional T residue in some RNAs). One
difference is that base pairing between G and U residues is allowed
in RNA (Fig. 8-23a and Fig. 8-24, inset) when complementary
sequences in two single strands of RNA pair with each other.
The paired strands in RNA duplexes (or
RNA-DNA duplexes) are antiparallel, as
in double-helical DNA. The predominant
double-stranded structure is an A-form
right-handed double helix. The B-form
double helix has not been observed for
RNA. Strands of RNA that are
precisely complementary over long
regions of sequence are uncommon.
Breaks in the regular A-form RNA
double helix caused by mismatched or
unmatched bases in one or both strands
are common and result in bulges or
internal loops (Fig. 8-23a). Hairpin
loops form between nearly self-
complementary (palindromic) sequences.
RNA Secondary Structures (II)
The potential for base-paired helical
segments in many RNAs is extensive, and the
resulting hairpins are the most common type
of secondary structure in RNA. The
extensive secondary structure of an RNA is
illustrated for the M1 RNA component of the
enzyme RNase P of E. coli, that functions in
the processing of tRNA (Fig. 8-24). This
enzyme also contains a protein component
(not shown). The two brackets in the figure
indicate additional complementary sequences
that may be paired in the three dimensional
structure. The blue dots indicate non-
Watson-Crick G/U base pairs (see inset).
Note that G/U base pairs are only allowed
when presynthesized RNA strands anneal
together, and are not inserted by RNA
polymerases into an RNA strand across from
a G in a DNA template.
 Three-dimensional Structure in RNA
Extensive secondary
structures, as illustrated for
the M1 RNA, act as starting
points for the folding of an
RNA molecule into its precise
three-dimensional structure.
Other contributions are made
by hydrogen bonds that are
not part of standard
Watson-Crick base pairs.
These include bonds involving
the 2’-hydroxyl group of
ribose, and the oxygens of
ribose phosphodiester bonds
(Fig. 8-25a). The three-
dimensional structures of
phenylalanine tRNA of yeast,
a hammerhead ribozyme from
certain plant viruses, and the
self-splicing intron of a
mRNA from the ciliated
protozoan, Tetrahymena
thermophila are shown in the
figure.