Nucleotides and Nucleic Acids

• Some Basics
• Nucleic Acid Structure
• Nucleic Acid Chemistry
• Other Functions of Nucleo-
tides

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 ge-
nomic 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 partici-
pate 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 phos-
phates.
• In the absence of the phosphate group(s), the molecule is called a nu-
cleoside.
• The nitrogenous bases are derivatives of pyrimidine and purine. The
numbering of the ring atoms of pyrimidines and purines is shown be-
low. The numbering of the pentose rings follows the convention out-
lined, 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).
• 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 ma-
nure) and thymine was first isolated from thymus tissue.
Nomenclature of Nucleosides & Nu-
cleotides
The names of the nucleosides and nucleotides containing the
five common bases are listed below.
 The Pentoses of Nucleotides
• Nucleotides have two kinds of pentoses.
• The recurring deoxyribonucleotide units of DNA contain 2’-deoxy-D-ri-
bose, 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 alde-
hyde form of D-ribose in solution is illustrated below.
• 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 .
• 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.
• All nucleotides are shown in their free form at pH 7.0. The deoxyri-
bonucleotide 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 deoxyri-
bonucleotides 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 (ribonu-
cleoside 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.
• In DNA, the most common of these are
methylated forms of the major bases
(Panel a). Minor bases of many types oc-
cur 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 sub-
stituent by its number, e.g., 5-methylcy-
tidine.
• The convention changes when the sub-
stituent 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 at-
tached 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. For example, ri-
bonucleoside 2’,3’-cyclic monophosphates are isolatable inter-
mediates, 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 Cova-
lent 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 nucleo-
tide unit is joined to the 3’-hydroxyl group of the
next, creating a phosphodiester linkage.
• Thus, the covalent backbones of nucleic acids
consist of alternating phosphate and pentose
residues, and the nitrogenous bases may be re-
garded as side groups joined to the backbone at
regular intervals.
• The backbones of both DNA and RNA are hy-
drophilic.
• 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 pro-
teins, 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.
 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. 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 pentadeoxyribonu-
cleotide 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 fig-
ure 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 tau-
tomeric forms depending on the pH. Uracil, for example, occurs
in lactam, lactim, and double lactim forms depending on the pH.
• Certain tautomeric forms predominate at neutral pH, and these
are the structures shown for the five common purines and pyrim-
idines 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.
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, car-
bonyl 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 Wat-
son and Crick in 1953, in which A bonds specifically to T (or U) and G
bonds to C.
• These two types of base pairs predominate in double-stranded DNA and
RNA, and the tautomers shown in 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
A model for the structure of DNA (I)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 composi-
tion of DNA and the x-ray diffraction properties of DNA fibers.
Most importantly, x-ray diffraction studies of DNA fibers per-
formed by Rosalind Franklin and Maurice Wilkins (Fig. 8-12)
showed that DNA molecules are helical and exhibit two periodici-
ties 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
the sum ofequals
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
In DNA, Watson and Crick proposed(II)
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 orienta-
tion, and this was later proven to be
true. The hydrophilic backbones of al-
ternating deoxyribose and phosphate
groups are on the outside of the helix
facing the surrounding water. The fu-
ranose 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
Base stacking accounts for the 3.4 (III)
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 modi-
fied 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 in-
side the double helix. Finally, the offset
pairing of the two strands creates a
major and a minor groove on the sur-
face of the duplex. It should be noted
that the double helix not only is stabi-
lized by Watson-Crick base pairing be-
tween 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 bio-
chemical evidence.
Double-helical Strand Complementar-
ity
• The two antiparallel chains of double-helical
DNA are not identical in either base se-
quence or composition.
• Instead, they are complementary to one
another. Wherever adenine occurs in one
chain, thymine occurs in the other. Simi-
larly, guanine occurs opposite cytosine in
the two chains.
Watson-Crick Model for DNA Replica-
The model for DNA structure tion
immedi-
ately suggested to Watson and Crick a
mechanism for the transmission of ge-
netic information. The essential feature
of the model is the complementarity of
the two DNA strands in the double he-
lix. As Watson and Crick were able to
see, well before confirmatory data be-
came available, this structure could log-
ically be replicated by separating the
two strands, and synthesizing a com-
plementary strand for each. Because
nucleotides in each strand are joined in
a sequence specified by the base-pair-
ing 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 phospho-
deoxyribose backbone, and thermal fluctuation can produce bending, stretch-
ing, 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, ro-
tation about the contiguous bonds that make up the phosphodeoxyribose back-
bone, and free rotation about the C-1’-N-glycosyl bond.
• Because of steric constraints, purines in purine nucleotides are restricted to two
stable conformations with respect to deoxyribose, called syn and anti. Pyrim-
idines generally are restricted to the anti conformation because of steric inter-
ference 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 struc-
ture for a random-sequence DNA
molecule under physiological condi-
tions and is therefore the standard
structural reference in any study of
the properties of DNA. Two structural
variants that have been well charac-
terized in crystal structures are the A-
and Z-forms of DNA (Fig. 8-17). The
properties of these forms are summa-
rized in the Fig. 8-17 table (next
slide). In general, A-DNA is a dehy-
drated form of DNA that may not oc-
cur in cells. A similar type of struc-
ture 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. Cer-
tain 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, alter-
nating 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 metab-
olism of the DNA segments in their immediate vicinity. A rather
common type of such DNA sequence is a palindrome. A palin-
drome is a word, phrase, or sentence that is spelled identically
read either forward or backward. A example is the word ROTA-
TOR. 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 se-
quence 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 cruci-
form (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 thou-
sands. 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 pro-
tonated 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 re-
ferred to as Hoogsteen positions, and the non-Watson-Crick pair-
ing is called Hoogsteen pairing after their discoverer, Karst Hoog-
steen. 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 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 are
most stable at low pH because the C/G.C+ triplet requires a proto-
nated 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. 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.
 Intro. to RNA Structure
As in the case of protein structure, it is sometimes useful to de-
scribe 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 chromo-
somes within eukaryotic chromatin and the bacterial nucleoid, or
the elaborate folding of large tRNA or rRNA molecules, is gener-
ally 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). 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 al-
ways single-stranded RNA. The single
strand tends to assume a right-handed he-
lical conformation dominated by base-
stacking interactions. In this structure the
bases are shown in yellow, the phospho-
rous atoms in orange, and the ribose and
phosphate oxygens in green. Base stacking
interactions are stronger between two
purines than between a purine and pyrimi-
dine 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 pro-
duce more complex secondary structures. 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 dif-
ference is that base pairing between G and U residues is allowed in
RNA when complementary sequences in two single strands of RNA
pair paired
The with each other.
strands in RNA duplexes (or
RNA-DNA duplexes) are antiparallel, as
in double-helical DNA. The predomi-
nant double-stranded structure is an A-
form right-handed double helix. The B-
form double helix has not been ob-
served for RNA. Strands of RNA that
are precisely complementary over long
regions of sequence are uncommon.
Breaks in the regular A-form RNA dou-
ble 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) se-
quences.
RNA Secondary Structures
(II)potential for base-paired helical seg-
The
ments in many RNAs is extensive, and the
resulting hairpins are the most common
type of secondary structure in RNA. The ex-
tensive secondary structure of an RNA is il-
lustrated for the M1 RNA component of the
enzyme RNase P of E. coli, that functions in
the processing of tRNA. This enzyme also
contains a protein component (not shown).
The two brackets in the figure indicate addi-
tional complementary sequences that may
be paired in the three dimensional struc-
ture. 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 to-
gether, and are not inserted by RNA poly-
merases into an RNA strand across from a G
in a DNA template.
 Three-dimensional Structure in RNA
Extensive secondary struc-
tures, as illustrated for the
M1 RNA, act as starting
points for the folding of an
RNA molecule into its pre-
cise three-dimensional
structure. Other contribu-
tions 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 phospho-
diester bonds. The three-di-
mensional 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.