Nucleotides

NUCLEOSIDES, NUCLEOTIDES, DNA,

RNA
 PURINES, PYRIMIDINES, NUCLEOSIDES,
& NUCLEOTIDES
• Purines and pyrimidines are
nitrogen-containing heterocycles,
cyclic compounds whose rings
contain both carbon and other
elements (hetero atoms).
• Note that the smaller pyrimidine
has the longer name and the
larger purine the shorter name
and that their six-atom rings are
numbered in opposite directions.
 Nucleosides & Nucleotides
• Nucleosides are derivatives of
purines and pyrimidines that
have a sugar linked to a ring
nitrogen. Numerals with a
prime (eg, 2′ or 3′) distinguish
atoms of the sugar from those
of the heterocyclic base.
• The sugar in ribonucleosides
is D-ribose, and in deoxyribo
nucleosides it is 2-deoxy-D-
ribose. The sugar is linked to
the heterocyclic base via a -N-
glycosidic bond, almost
always to N-1 of a pyrimidine
or to N-9 of a purine.
 Nucleotides
• Nucleotides have three characteristic components:
• (1) a nitrogenous (nitrogen-containing) base,
• (2) a pentose, and
• (3) a phosphate (Fig).
• The molecule without the phosphate group is called a
nucleoside.
• The nitrogenous bases are derivatives of two parent
compounds, pyrimidine and purine.
 BIOMEDICAL IMPORTANCE

• Nucleotides—the monomer units or building blocks of

nucleic acids—serve multiple additional functions.
• They form a part of many coenzymes and serve as donors
of
• Phosphoryl groups (eg, ATP or GTP), of sugars (eg, UDP- or
GDP-sugars), or of lipid (eg, CDP-acylglycerol).
• Regulatory nucleotides include the second messengers
cAMP and cGMP, the control by ADP of oxidative
phosphorylation, and allosteric regulation of enzyme
activity by ATP, AMP, and CTP.
• Synthetic purine and pyrimidine analogs that contain
halogens, thiols, or additional nitrogen are employed for
chemotherapy of cancer and AIDS and as suppressors of
the immune response during organ transplantation.
 Nucleic Acid Structure & Function
• BIOMEDICAL IMPORTANCE
• The discovery that genetic information is coded
along the length of a polymeric molecule
composed of only four types of monomeric units
was one of the major scientific achievements of
the twentieth century.
• This polymeric molecule, DNA, is the chemical
basis of heredity and is organized into genes, the
fundamental units of genetic information.
• The basic information pathway—ie, DNA directs the
synthesis of RNA, which in turn directs protein synthesis
—has been elucidated.
• Genes do not function autonomously; their replication
and function are controlled by various gene products,
often in collaboration with components of various signal
transduction pathways.
• Knowledge of the structure and function of nucleic acids
is essential in understanding genetics and many aspects
of pathophysiology as well as the genetic basis of
disease
 DNA Contains Four Deoxynucleotides
• The chemical nature of the
monomeric deoxynucleotide
units of DNA are
deoxyadenylate,
deoxyguanylate,
deoxycytidylate, and
thymidylate
• These monomeric units of DNA
are held in polymeric form by
3′,5′-phosphodiester bridges
constituting a single strand.
• The polymer possesses a
polarity; one end has a 5′-
hydroxyl or phosphate terminal
while the other has a 3′-
phosphate or hydroxyl terminal.
Nucleic Acid Structure

• Watson-Crick model for the

structure of DNA.
• The original model proposed by
Watson and Crick had 10 base
pairs, or 34 Å (3.4 nm), per turn of
the helix;
• subsequent measurements
revealed 10.5 base pairs, or 36 Å
(3.6 nm), per turn.
• (a) Schematic representation,
showing dimensions of the helix.
• (b) Stick representation showing
• the backbone and stacking of the
bases.
• (c) Space-filling model
• The discovery of the structure of DNA by Watson and Crick in 1953 was a
momentous event in science, an event that gave rise to entirely new
disciplines.
• In 1953 Watson and Crick postulated a three dimensional model of DNA
structure that accounted for all the available data. It consists of two helical
DNA chains wound around the same axis to form a right handed double
helix.
• The hydrophilic backbones of alternating deoxyribose and phosphate groups
are on the outside of the double helix, facing the surrounding water.
• The purine and pyrimidine bases of both strands are stacked inside.
• The offset pairing of
• the two strands creates a major groove and minor groove on the surface of
the duplex.
• Each nucleotide base of one strand is paired in the same plane with a base of
the other strand. Watson and Crick found that the hydrogen-bonded base
pairs, G with C and A with T.
• Complemetry base pairing in DNA proves Chargaff’s rule that in any DNA, G
= C and A = T.
• It is important to note that three hydrogen bonds can form between G and
• The two DNA strands are antiparallel , that is one run in 3’ to 5’
direction while the other run in 5’ to 3’ direction.
• stacked bases inside the double
• helix would be 3.4 Å apart; the secondary repeat distance
• of about 34 Å was accounted for by the presence
• of 10 base pairs in each complete turn of the double helix.
• The stacked bases inside the double helix would be 3.4 Å apart;
each turn of DNA is about 34 Å accounted for by the presence
of 10 base pairs in each complete turn of the double helix.
• The two antiparallel polynucleotide chains of double-helical
DNA are not identical in either base sequence or composition.
Instead they are complementary to each other. Wherever
adenine occurs in one chain, thymine is found in the other;
similarly, wherever guanine occurs in one chain, cytosine is
found in the other.
• The DNA double
helix, or duplex, is
held together by two
forces, as described
earlier: hydrogen
bonding between
complementary base
pairs (Fig) and base-
stacking interactions.
 Ribonucleic acid (RNA)
• Ribonucleic acid (RNA) is a polymer of purine and
pyrimidine ribonucleotides linked together by 3′,5′-
phosphodiester bridges. Although sharing many
features with DNA, RNA possesses several specific
differences:
• (1) In RNA, the sugar moiety to which the phosphates
and purine and pyrimidine bases are attached is ribose
rather than the 2′-deoxyribose of DNA.
• (2) The pyrimidine components of RNA differ from
those of DNA. Although RNA contains the
ribonucleotides of adenine, guanine, and cytosine, it
does not possess thymine except in the rare case.
Instead of thymine, RNA contains the ribonucleotide of
uracil .
 contd

• (3) RNA exists as a single strand, whereas DNA exists as a

double-stranded helical molecule. However, given the
proper complementary base sequence with opposite
polarity, the single strand of RNA is capable of folding back
on itself like a hairpin and thus acquiring double stranded
characteristics.
• (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 equal its cytosine content, nor does its
adenine content necessarily equal its uracil content.
• (5) RNA can be hydrolyzed by alkali to 2′,3′ cyclic diesters of
the mononucleotides, compounds that cannot be formed
from alkali-treated DNA because of the absence of a 2 ′-
hydroxyl group. The alkali lability of RNA is useful both
diagnostically and analytically.
 RNA Are Involved in Protein Synthesis
• Those cytoplasmic RNA molecules that serve as templates
for protein synthesis (ie, that transfer genetic information
from DNA to the protein-synthesizing machinery) are
designated messenger RNAs, or mRNAs.
• Many other cytoplasmic RNA molecules (ribosomal RNAs;
rRNAs) have structural roles wherein they contribute to
the formation and function of ribosomes (the organellar
machinery for protein synthesis) or
• serve as adapter molecules (transfer RNAs; tRNAs) 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 involves the cleavage of
a nucleic acid.
• A segment of a ribonucleic
acid (RNA) molecule in
which the purine and
pyrimidine bases—
• guanine (G), cytosine (C),
uracil (U), and adenine (A)
—are held together by
phosphodiester bonds
between ribosyl moieties
attached to the
nucleobases by N-
glycosidic bonds.
• Note that the polymer has
a polarity as indicated by
the labeled 3′- and 5′-
attached phosphates.
 MESSENGER RNA (mRNA)
• This class is the most heterogeneous in size and stability.
• All members of the class function as messengers
conveying the information in a gene to the protein
synthesizing machinery, where each serves as a template
on which a specific sequence of amino acids is
polymerized to form a specific protein molecule, the
ultimate gene product
• Messenger RNAs, particularly in eukaryotes, have some
unique chemical characteristics. The 5′ terminal of mRNA
is “capped” by a 7-methylguanosine triphosphate that is
linked to an adjacent 2′-O-methyl ribonucleoside at its 5′-
hydroxyl through the three phosphates.
• The cap is involved in the recognition of mRNA by
the translating machinery, and it probably helps
stabilize the mRNA by preventing the attack of 5 ′-
exonucleases.
• The protein-synthesizing machinery begins
translating the mRNA into proteins beginning
downstream of the 5′ or capped terminal.
• The other end of most mRNA molecules, the 3 ′-
hydroxyl terminal, has an attached polymer of
adenylate residues 20–250 nucleotides in length.
• The specific function of the poly(A) “tail” at the 3 ′-
hydroxyl terminal of mRNAs is not fully understood,
but it seems that it maintains the intracellular
stability of the specific mRNA by preventing the
attack of 3′-exonucleases.
The relationship between the sequences of an RNA
transcript and its gene, in which the coding and
template strands are shown with their polarities.
The RNA transcript with a 5′ to 3′ polarity is
complementary to the template strand with its 3′
to 5′ polarity. Note that the sequence in the RNA
transcript and its polarity is the same as that in the
coding strand, except that the U of the transcript
replaces the T of the gene.
The expression of genetic information in DNA into the form of an
mRNA transcript. This is subsequently translated by ribosomes into a
specific protein molecule.
• In mammalian nuclei, the immediate products of
gene transcription constitute a fourth class of RNA
molecules.
• These nuclear RNA molecules are very heterogeneous
in size and are quite large. The heterogeneous nuclear
RNA (hnRNA) molecules may have a molecular weight
in excess of 107, whereas the molecular
• weight of mRNA molecules is generally less than 2 ×
106. hnRNA molecules are processed to generate the
mRNA molecules which then enter the cytoplasm to
serve as templates for protein synthesis.
 TRANSFER RNA (tRNA)
• tRNA molecules vary in length from 74 to 95
nucleotides. They also are generated by nuclear
processing of a precursor molecule. The tRNA
molecules serve as adapters for the translation of the
information in the sequence of nucleotides of the
mRNA into specific amino acids.
• There are at least 20 species of tRNA molecules in every
cell, at least one (and often several) corresponding to
each of the 20 amino acids 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—ie, the nucleotide sequence—of
all tRNA molecules allows extensive folding and
intrastrand complementarity to generate a secondary
structure that appears like a cloverleaf.
All tRNA molecules contain four main arms. The
acceptor arm terminates in the nucleotides CpCpAOH.
These three nucleotides are added post
transcriptionally . The tRNA-appropriate amino acid is
attached to the 3′-OH group of the A moiety of the
acceptor arm.
The D, TΨC, and extra arms help define a specific tRNA.
Typical aminoacyl tRNA in which the amino acid (aa) is attached to the
3′ CCA terminal. The anticodon, TΨC, and dihydrouracil (D) arms are
indicated, as are the positions of the intramolecular hydrogen bonding
between these base pairs.
 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. In 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 2.8 × 106 (60S)
and a smaller subunit with a molecular weight of 1.4 × 106 (40S).
• The 60S subunit contains a 5S ribosomal RNA (rRNA), a 5.8S
rRNA, and a 28S rRNA; there are also probably more than 50
specific polypeptides.
• The 40S subunit is smaller and contains a single 18S rRNA and
approximately 30 distinct polypeptide chains.
• All of the ribosomal RNA molecules except the 5S rRNA are
processed from a single 45S precursor RNA molecule in the
nucleolus.
• 5S rRNA is independently transcribed. The highly methylated
ribosomal RNA molecules are packaged in the nucleolus with
the specific ribosomal proteins.
• In the cytoplasm, the ribosomes remain quite stable and
capable of many translation cycles.
• The functions of the ribosomal RNA molecules in the ribosomal
particle are not fully understood, but they are necessary for
ribosomal assembly and seem to play key roles in the binding of
mRNA to ribosomes and its translation.
• Recent studies suggest that an rRNA component performs the
peptidyl transferase activity and thus is an enzyme (a ribozyme).
 RNA Synthesis
• The synthesis of an RNA molecule from DNA is a
complex process involving one of the group of RNA
polymerase enzymes and a number of associated
proteins.
• The general steps required to synthesize the primary
transcript are initiation, elongation, and termination.
Most is known about initiation. A number of DNA
regions (generally located upstream from the initiation
site) and protein factors that bind to these sequences
to regulate the initiation of transcription have been
identified.
 RNA IS SYNTHESIZED FROM A DNA TEMPLATE
• The strand that is transcribed or copied into an RNA molecule is
referred to as the template strand of the DNA. The other DNA
strand is frequently referred to as the coding strand of that gene.
• It is called this because, with the exception of T for U changes, it
corresponds exactly to the sequence of the primary transcript,
which encodes the protein product of the gene. In the case of a
double-stranded DNA molecule containing many genes, the
template strand for each gene will not necessarily be the same
strand of the DNA double helix. Thus, a given strand of a double-
stranded DNA molecule will serve as the template strand for some
genes and the coding strand of other genes. Note that the
nucleotide sequence of an RNA transcript will be the same (except
for U replacing T) as that of the coding strand. The information in
the template strand is read out in the 3 ′ to 5 ′ direction.
 DNA-Dependent RNA Polymerase Initiates Transcription at a
Distinct Site, the Promoter

• DNA-dependent RNA polymerase is the enzyme responsible

for the polymerization of ribonucleotides into a sequence
complementary to the template strand of the gene. The
enzyme attaches at a specific site—the promoter—on the
template strand. This is followed by initiation of RNA
synthesis at the starting point, and the process continues until
a termination sequence is reached.
• A transcription unit is defined as that region of DNA that
includes the signals for transcription initiation, elongation,
and termination.
• The RNA product, which is synthesized in the 5 ′ to 3 ′
direction, is the primary transcript.
• RNA polymerase (RNAP) catalyzes
the polymerization of
ribonucleotides into an RNA
sequence that is complementary to
the template strand of the gene.
• The RNA transcript has the same
polarity (5′ to 3′) as the coding
strand but contains U rather than T.
E coli RNAP consists of a core
complex of two α subunits and two
β subunits (β and β′). The
holoenzyme contains the σ subunit
bound to the α2ββ′ core assembly.
The ω subunit is not shown. The
transcription “bubble” is an
approximately 20-bp area of melted
DNA, and the entire complex covers
30–75 bp, depending on the
conformation of RNAP.
 Stages of transcription
• The transcription cycle in bacteria.
• Bacterial RNA transcription is described in four steps:
• (1) Template binding: RNA polymerase (RNAP) binds to DNA and
locates a promoter (P) melts the two DNA strands to form a
preinitiation complex (PIC).
• (2) Chain initiation: RNAP holoenzyme (core + one of multiple sigma
factors) catalyzes the coupling of the first base (usually ATP or GTP) to
a second ribonucleoside triphosphate to form a dinucleotide.
• (3) Chain elongation: Successive residues are added to the 3′-OH
terminus of the nascent RNA molecule.
• (4) Chain termination and release: The completed RNA chain and
RNAP are released from the template. The RNAP holoenzyme are-
forms, finds a promoter, and the cycle is repeated.
 Protein Synthesis & the Genetic Code
• BIOMEDICAL IMPORTANCE
• The letters A, G, T, and C correspond to the nucleotides found in
DNA. They are organized into three letter code words called
codons, and the collection of these codons makes up the genetic
code.
• It was impossible to understand protein synthesis—or to explain
mutations—before the genetic code was elucidated.
• The code provides a foundation for explaining the way in which
protein defects may cause genetic disease and for the diagnosis
and perhaps the treatment of these disorders.
• In addition, the pathophysiology of many viral infections is related
to the ability of these agents to disrupt host cell protein synthesis.
• Many antibacterial agents are effective because they selectively
disrupt protein synthesis in the invading bacterial cell but do not
affect protein synthesis in eukaryotic cells.
 GENETIC INFORMATION FLOWS FROM DNA TO RNA TO
PROTEIN
• The genetic information within the nucleotide sequence of
DNA is transcribed in the nucleus into the specific nucleotide
sequence of an RNA molecule. Several different classes of RNA
combine to direct the synthesis of proteins.
• In higher eukaryotic cells, the primary transcript is much larger
than the mature mRNA. The large mRNA precursors contain
coding regions (exons) that will form the mature mRNA and
long intervening sequences (introns) that separate the exons.
• The hnRNA is processed within the nucleus, and the introns,
which often make up much more of this RNA than the exons,
are removed.
• Exons are spliced together to form mature mRNA, which is
transported to the cytoplasm, where it is translated into
protein.
• The cell must possess the machinery necessary to translate information
accurately and efficiently from the nucleotide sequence of an mRNA into the
sequence of amino acids of the corresponding specific protein.
• Clarification of our understanding of this process, which is termed translation,
awaited deciphering of the genetic code.
• It was realized early that mRNA molecules themselves have no affinity for amino
acids and, therefore, that the translation of the information in the mRNA
nucleotide sequence into the amino acid sequence of a protein requires an
intermediate adapter molecule. This adapter molecule must recognize a specific
nucleotide sequence on the one hand as well as a specific amino acid on the
other.
• With such an adapter molecule, the cell can direct a specific amino acid into the
proper sequential position of a protein during its synthesis as dictated by the
nucleotide sequence of the specific mRNA. In fact, the functional groups of the
amino acids do not themselves actually come into contact with the mRNA
template.
 GENETIC CODE

• Twenty different amino acids are required for the

synthesis of the cellular complement of proteins; thus,
there must be at least 20 distinct codons that make up
the genetic code.
• Since there are only four different nucleotides in mRNA,
each codon must consist of more than a single purine or
pyrimidine nucleotide.
• Codons consisting of two nucleotides each could provide
for only 16 (42) specific codons, whereas codons of three
nucleotides could provide 64 (43) specific codons.
• It is now known that each codon consists of a sequence of
three nucleotides; ie, it is a triplet code.
 GENETIC CODE
• 1-The terms first, second, and
third nucleotide refer to the
individual nucleotides of a triplet
codon. U, uridine nucleotide; C,
cytosine nucleotide; A, adenine
nucleotide; G, guanine
nucleotide; Term, chain
terminator codon. AUG, which
codes for Met, serves as the
initiator codon in mammalian
cells and encodes for internal
methionines in a protein.
• 2-In mammalian mitochondria,
AUA codes for Met and UGA for
Trp, and AGA and AGG serve as
chain terminators.
 THE GENETIC CODE IS DEGENERATE, UNAMBIGUOUS, NONOVERLAPPING,
WITHOUT PUNCTUATION, & UNIVERSAL
• Three of the 64 possible codons do not code for specific amino acids; these have
been termed nonsense codons.
• These nonsense codons are utilized in the cell as termination signals; they specify
where the polymerization of amino acids into a protein molecule is to stop.
• The remaining 61 codons code for 20 amino acids. Thus, there must be
“degeneracy” in the genetic code—ie, multiple codons must decode the same
amino acid. Some amino acids are encoded by several codons; for example, six
different codons specify serine. Other amino acids, such as methionine and
tryptophan, have a single codon.
• However, for any specific codon, only a single amino acid is indicated; the genetic
code is unambiguous—ie, given a specific codon, only a single amino acid is
indicated. The distinction between ambiguity and degeneracy is an important
concept.
• The reading of the genetic code during the process of protein synthesis does not
involve any overlap of codons. Thus, the genetic code is non overlapping.
• Furthermore, once the reading is commenced at a specific codon, there is no
punctuation between codons, and the message is read in a continuing sequence
of nucleotide triplets until a translation stop codon is reached.
• The genetic code is universal.
 PROTEIN SYNTHESIS
• The ribosome serve as the machinery on which the
mRNA nucleotide sequence is translated into the
sequence of amino acids of the specified protein.
This process is known as translation.
• The message on mRNA is read from 5′ to 3′,
concluding with the formation of the carboxyl
terminal of the protein.
• In eukaryotic organisms, the process of translation
occurs in the cytoplasm.
• Like transcription, protein synthesis can be described
in three phases: initiation, elongation, & termination
 Initiation Involves Several Protein-RNA Complexes
• Initiation of protein synthesis requires that an mRNA molecule be
selected for translation by a ribosome.
• Once the mRNA binds to the ribosome, the latter finds the correct
reading frame on the mRNA, and translation begins.
• This process involves tRNA, rRNA, mRNA, and at least ten
eukaryotic initiation factors (eIFs). Also involved are GTP, ATP, and
amino acids.
• Initiation can be divided into four steps:
• (1) dissociation of the ribosome into its 40S and 60S subunits;
• (2) binding of a ternary complex consisting of met-tRNAi, GTP, and
eIF-2 to the 40S ribosome to form a preinitiation complex;
• (3) binding of mRNA to the 40S preinitiation complex to form a
43S initiation complex; and
• (4) combination of the 43S initiation complex with the 60S
ribosomal subunit to form the 80S initiation complex.
 Elongation Also Is a Multistep Process
• Elongation is a cyclic process on the ribosome in which one
amino acid at a time is added to the nascent peptide chain.
• The peptide sequence is determined by the ordern of the
codons in the mRNA.
• Elongation involves several steps catalyzed by proteins
called elongation factors (EFs).
• These steps are
• (1) binding of aminoacyl-tRNA to the A site,
• (2) peptide bond formation, and
• (3) translocation.
Diagrammatic representation
of the peptide elongation
process of protein synthesis.
The small circles labeled n − 1,
n, n + 1, etc, represent the
amino acid residues of the
newly formed protein
molecule.
EFIA and EF2 represent
elongation factors 1 and 2,
respectively.
The peptidyl-tRNA and
aminoacyl-tRNA sites on the
ribosome are represented by P
site and A site, respectively.
Termination Occurs When a Stop Codon Is Recognized

• In comparison to initiation and elongation, termination is a relatively simple

process. After multiple cycles of elongation culminating in polymerization of
the specific amino acids into a protein molecule, the stop or terminating
codon of mRNA (UAA, UAG, UGA) appears in the A site.
• Normally, there is no tRNA with an anticodon capable of recognizing such a
termination signal.
• Releasing factor RF1 recognizes that a stop codon resides in the A site. RF1
is bound by a complex consisting of releasing factor RF3 with bound GTP.
This complex, with the peptidyl transferase, promotes hydrolysis of the
bond between the peptide and the tRNA occupying the P site. Thus, a water
molecule rather than an amino acid is added.
• This hydrolysis releases the protein and the tRNA from the P site. Upon
hydrolysis and release, the 80S ribosome dissociates into its 40S and 60S
subunits, which are then recycled