Download The nucleotides

The nucleotides
• BIOMEDICAL IMPORTANCE
1-Serving as precursors of nucleic acids purine and pyrimidine nucleotides
2- When linked to vitamins or vitamin derivatives, nucleotides form a portion of
many coenzymes (NAD).
3- nucleoside tri- and diphosphates such as ATP and ADP are the principal players
in the energy transductions.
4- The cyclic nucleotides cAMP and cGMP serve as the second messengers in
hormonally regulated events.
5- Use of synthetic purine and pyrimidine analogs that contain halogens, thiols, or
additional nitrogen atoms in the chemotherapy of cancer and AIDS, and as
suppressors of the immune response during organ transplantation.
Purines & Pyrimidines :are nitrogen-containing heterocycles, structures that
contain, in addition to carbon, other atoms such as nitrogen. The smaller
pyrimidine molecule has the longer name and the larger purine molecule the
shorter name, and that their six-atom rings are numbered in opposite directions
(Figure1).
chemistry of purines ,pyrimidines , nucleosides &
nucleotides
• Both DNA and RNA contain the same
purine bases: adenine (A) and
guanine (G).and contain the
pyrimidine cytosine (C), but they
differ in their second pyrimidine
base: DNA contains thymine (T),
whereas RNA contains uracil (U).
• T and U differ by only one methyl
group, which is present on T but
absent on U
Nucleosides Are N -Glycosides
Nucleosides are derivatives of purines and pyrimidines that have a sugar linked to a
ring nitrogen of a purine or pyrimidine. The sugar in ribonucleosides is D-ribose, and
in deoxyribonucleosides is 2-deoxy-D-ribose. Both sugars are linked to the heterocycle
by a -N-glycosidic bond, almost always to the N-1 of a pyrimidine or to N-9 of a
purine.
The ribonucleosides of A, G, C, and U are named adenosine, Guanosine, cytidine, and
uridine, respectively. The deoxyribonucleoside of A, G, C, and T have the added prefix,
"deoxy-", for example deoxyadenosine .
Nucleotides Are Phosphorylated Nucleosides
Mononucleotides are nucleosides with a phosphoryl group esterified to a
hydroxyl group of the sugar. The 5'-nucleotides are nucleosides with a
phosphoryl group on the 5'-hydroxyl group of the sugar (nucleoside 5'phosphate or a 5'-nucleotide).
If one phosphate group is attached to the
5'-carbon of the pentose →nucleoside
monophosphate (NMP:AMP or CMP).
a second or third phosphate is added to
the nucleoside, a nucleoside diphosphate
(eg. ADP) or triphosphate (eg.ATP) results.
The second and third phosphates are each
connected to the nucleotide by a
"high-energy" bond. [The phosphate groups
are responsible for the negative charges
associated with nucleotides, and cause DNA
and RNA to be referred to as "nucleic acids."]
Heterocyclic N -Glycosides Exist as Syn
and Anti Conformers
• In nucleosides or nucleotides there is no
freedom of rotation about the -Nglycosidic bond. Both therefore exist as
non interconvertible syn or anti
conformers .
• syn and anti conformers can only be
interconverted by cleavage and
reformation of the glycosidic bond. Both
syn and anti conformers occur in nature,
but the anti conformers predominate.
• Modification of Polynucleotides
Small quantities of additional purines and
pyrimidines occur in DNA and RNAs. Examples
include
5-methylcytosine of bacterial and human DNA.
5-hydroxymethylcytosine of bacterial and viral
nucleic acids
DNA & RNA ARE POLYNUCLEOTIDES
The 5'-phosphoryl group of a mononucleotide can esterify a hydroxyl
group, forming a phosphodiester. Most commonly, this hydroxyl group
is the 3'-OH of the pentose of a second nucleotide. This forms a
dinucleotide in which the pentose moieties are linked by a 3',5'phosphodiester bond to form the "backbone" of RNA and DNA.
Nucleic acids
• Nucleic acids are required for the storage and expression
of genetic information. There are two types of nucleic
acids: deoxyribonucleic acid (DNA) and ribonucleic
acid.(RNA)
• DNA, the storehouse of genetic information, is present not
only in chromosomes in the nucleus of eukaryotic
organisms, but also in mitochondria and the chloroplasts
of plants. Prokaryotic cells, which lack nuclei, have a single
chromosome, but may also contain DNA in the form of
plasmids.
• The genetic information found in DNA is copied and
transmitted to daughter cells through DNA replication.
• Transcription (RNA synthesis) is the first stage in the
expression of genetic information.
• The code contained in the nucleotide sequence of
messenger RNA molecules is translated (protein synthesis),
thus completing gene expression.
• This flow of information from DNA to RNA to protein is
termed the "central dogma
STRUCTURE OF DNA
• DNA is contains many deoxyribonucleotides covalently linked by
bonds. With the exception of a few viruses that contain singlestranded DNA, DNA exists as a double-stranded molecule, in which
the two strands wind around each other, forming a double helix.
• eukaryotic cells, DNA is found associated with various types of
proteins (known collectively as nucleoprotein) present in the
nucleus, whereas in prokaryotes, the protein-DNA complex is
present in the nucleoid.
• Phosphodiester bonds join the 5'-hydroxyl group of the
deoxypentose of one nucleotide to the 3'-hydroxyl group of the
deoxypentose of an adjacent nucleotide through a phosphate group
• The resulting long, unbranched chain has polarity, with both a 5'end (free phosphate) and a 3'-end (free hydroxyl) that are not
attached to other nucleotides. The bases located along the resulting
deoxyribose-phosphate backbone are always written in sequence
from the 5'-end of the chain to the 3'-end
(5'-TACG-3').
Phosphodiester linkages between nucleotides (in DNA or RNA) can be
cleaved hydrolytically by chemicals, or hydrolyzed by a family of
nucleases: deoxyribonucleases for DNA and ribonucleases for RNA.
[endonucleases: cleave the nucleotide chain at positions in the interior
exo nucleases: cleave the chain only by removing individual nucleotides
from one of the two ends ]
Double helix
• the two chains are coiled around a common
Axis called the axis of symmetry. The chains
are paired in an antiparallel manner, the 5'-end
of one strand is paired with the3'-end of the
other strand
• the hydrophilic deoxyribose- Phosphate
backbone of each chain is on the outside of the
molecule, whereas the hydro phobic bases are
stacked inside. The overall structure resembles
a twisted ladder. The spatial relationship between the two
strands creates a major and a minor groove .
• Certain anticancer drugs, such as dactinomycin (actinomycin D), exert
their cytotoxic effect by intercalating into the narrow groove of the
DNA double helix, thus interfering with RNA and DNA synthesis.1]
Base pairing
• Base pairing: The bases of one strand of
DNA are paired with the bases of the second
strand, so that an adenine is always paired
with a thymine and a cytosine is always
paired with a guanine. Therefore, one
polynucleotide chain of the DNA double
helix is always the complement of the other.
• The specific base pairing in DNA leads to
Chargaff's Rules: In any sample of doublestranded DNA, the amount of adenine
equals the amount of thymine, the amount
of guanine equals the amount of cytosine,
and the total amount of purines equals the
total amount of pyrimidines.
• The base pairs are held together by
hydrogen bonds : two between A and T and
three between G and C. These hydrogen
bonds, plus the hydrophobic interactions
between the stacked bases, stabilize the
structure of the double helix.
Circular DNA molecules
Each chromosome in the nucleus of a eukaryote contains one long linear
molecule of double-stranded DNA, which is bound to a complex mixture
of proteins to form chromatin. Eukaryotes have also closed circular
DNA molecules in their mitochondria, as do plant chloroplasts.
A prokaryotic organism contains a single, double-stranded, supercoiled,
circular chromosome. In addition, most species of bacteria also contain
small, circular, extra chromosomal DNA molecules called plasmids.
Quadruple DNA G-quadruplexes (also known as G-tetrads or G4DNA) are nucleic acid sequences that are rich in guanine and are capable
of forming a four-stranded structure. Four guanine bases can associate
through hydrogen bonding to form a square planar structure called a
guanine tetrad, and two or more guanine tetrads can stack on top of each
other to form a G-quadruplex. The quadruplex structure is further
stabilized by the presence of a cation, especially potassium, which sits in
a central channel between each pair of tetrads .
• The formation of these
quadruplexes in telomeres
[consists of many repeats of the
sequenced(TTAGGG)]. has
been shown to decrease the
activity of the enzyme
telomerase which is responsible
for maintaining length of
telomeres and is involved in
around 85% of all cancers. This
is an active target of drug
discovery
•
DNA Exists in Relaxed & Supercoiled Forms
In some organisms such as bacteria, as well as
organelles such as mitochondria, the ends of the
DNA molecules are joined to create a closed circle
with no covalently free ends. Closed circles exist in
relaxed or supercoiled forms.
• Supercoils are introduced when a closed circle is
twisted around its own axis or when a linear
piece of duplex DNA, whose ends are fixed, is
twisted.
• Negative supercoils the molecule is twisted in
the direction opposite from the clockwise turns
of the right-handed double helix found in B-DNA.
• Enzymes that catalyze topologic changes of DNA
are called topoisomerases. Topoisomerases can
relax or insert supercoils, using ATP as an energy
source. Homologs of this enzymes are important
Sense and antisense
• A DNA sequence is called "sense" if its sequence is the
same as that of a messenger RNA copy that is translated
into protein.
• The sequence on the opposite strand is called the
"antisense" sequence.
• Both sense and antisense sequences can exist on different
parts of the same strand of DNA .
Separation of the two DNA strands in the double helix
• The two strands of the double helix separate when hydrogen bonds
between the paired bases are disrupted.
• Disruption can occur if the pH is altered (nucleotide bases ionize)
or if the solution is heated. [Phosphodiester bonds are not broken by
such treatment.]
• When DNA is heated, the temperature at which one half of the
helical structure is lost is defined as the melting temperature.
• The loss of helical structure in DNA, called denaturation.
• Because there are three hydrogen bonds between G and C but only
two between A and T, DNA that contains high concentrations of A
and T denatures at a lower temperature than G- and C-rich DNA
Under appropriate conditions,
• complementary DNA strands can reform the double helix by the
process called renaturation (or reannealing).
Chromatin is the chromosomal material in the
nuclei of eukaryotic
• Chromatin consists of very long doublestranded DNA molecules and a nearly equal
mass of rather small basic proteins termed
histones as well as a smaller amount of
nonhistone proteins (most of which are acidic
and larger than histones) and a small quantity
of RNA.
• The nonhistone proteins include enzymes
involved in DNA replication and repair, and the
proteins involved in RNA synthesis, processing,
and transport to the cytoplasm.
• Electron microscopic studies of chromatin have
demonstrated dense spherical particles called
nucleosomes, which are approximately 10 nm
in diameter and connected by DNA filaments .
• Nucleosomes are composed of DNA wound
around a collection of histone molecules
chromosomes possess a 2-fold symmetry, with the identical
duplicated sister chromatids connected at a centromere.
The centromere is an adenine-thymine (A–T)-rich region containing
repeated DNA
DNA IS ORGANIZED INTO CHROMOSOMES
• the kinetochore, provides the anchor for the
mitotic spindle.
•The ends of each chromosome contain structures
called telomeres. Telomeres consist of short TG-rich
repeats.
• Human telomeres have a variable number of
repeats of the sequence 5'-TTAGGG-3.
Telomerase, a multisubunit RNA-containing
complex , is the enzyme responsible for telomere
synthesis and thus for maintaining the length of
the telomere. Since telomere shortening has
been associated with both malignant
transformation and aging, telomerase has
become an attractive target for cancer
chemotherapy and drug development.
DNA SYNTHESIS & REPLICATION
In all cells, replication can occur only
from a single-stranded DNA (ssDNA)
template.
Steps Involved in DNA Replication
in Eukaryotes
1. Identification of the origins of
replication
2. Unwinding (denaturation) of
dsDNA to provide an ssDNA
template
3. Formation of the replication fork;
synthesis of RNA primer
4. Initiation of DNA synthesis and
elongation
5. Formation of replication bubbles
with ligation of the newly
synthesized DNA segments
A-origin of replication .
• prokaryotic organisms, begins at a single, unique nucleotide site .
• In eukaryotes, begins at multiple sites along the DNA helix .These sites
include a short sequence composed almost exclusively of AT base
pairs.
• B- Separation (Unwinding )of the two complementary DNA strands
In order for the two strands of DNA to be replicated, they must first
separate (or "melt"), at least in a small region, because the polymerases
use only single-stranded DNA as a template.
Proteins required for DNA strand separation :
a. dnaA protein: bind to specific nucleotide sequences at the origin of
replication. This causes the strands separate, forming single-stranded
DNA.
b. Single-stranded DNA-binding (SSB) proteins
1. keep the two strands of DNA separated,
2. protect the DNA from nucleases that cleave single-stranded DNA.
c. DNA helicases: These enzymes bind to single-stranded DNA near the
replication fork, and then move into the neighboring double-stranded
region, forcing the strands apart in effect, unwinding the double helix.
Formation of the Replication Fork
A replication fork consists of:
(1) DNA helicase unwinds a short segment of the parental duplex DNA.
(2) a primase initiates synthesis of an RNA molecule that is essential for
priming DNA synthesis.
(3) the DNA polymerase initiates nascent, daughter strand synthesis.
(4) SSBs bind to ssDNA and prevent premature reannealing of ssDNA to
dsDNA.
The DNA polymerases responsible for copying
the DNA templates are only able to "read" the
parental nucleotide sequences in the direction
3'-»5, and they synthesize the new DNA strands
in the 5'->3' .the two newly synthesized
stretches of nucleotide chains must grow in
opposite directions—one in the 5'->3' direction
toward the replication fork and one in the 5'>3' direction away from the replication fork .
1. Leading strand: The strand that is being
copied in the direction of the advancing
replication fork and is synthesized almost
continuously.
2. Lagging strand: The strand that is being copied in the direction away
from the replication fork is synthesized discontinuously, with small
fragments of DNA copied near the replication fork. These short stretches
of discontinuous DNA, termed Okazaki fragments, are eventually joined
to become a single ,continuous strand.
The DNA Polymerase Complex
A number of different DNA polymerase molecules engage in DNA
replication. These share three important properties:
1-chain elongation accounts for the rate (in nucleotides per second;
ntd/s) at which polymerization occurs.
2- processivity is an expression of the number of nucleotides added to
the nascent chain before the polymerase disengages from the template.
3- proofreading function identifies copying errors and corrects them.
Polymerase I (pol I) and II (pol II) are mostly involved in proofreading
and DNA repair.
Eukaryotic cells have a large number of additional DNA polymerases
In mammalian cells, the polymerase is capable of polymerizing at a rate
that is somewhat slower than the rate of polymerization of
deoxynucleotides by the bacterial DNA polymerase complex. This
reduced rate may result from interference by nucleosomes
STRUCTURE OF RNA :
There are three major types of RNA that participate
in the process of protein synthesis:
ribosomal RNA (rRNA), transfer RNA (tRNA), and
messenger RNA (mRNA)
A. Ribosomal RNA (rRNAs) are found in association
with several proteins serve as the sites for protein
synthesis . There are three distinct size species of
rRNA (23S, 16S, and 5S) in prokaryotic cells
• In the eukaryotic cytosol, there are four rRNA size
species (28S, 18S, 5.8S, and 5S).
Transfer RNAs (tRNAs), the smallest of the three
major species of RNA molecules (4S), There is at
least one specific type of tRNA molecule for each
of the twenty amino acids commonly found in
proteins. Each tRNA serves as an "adaptor"
molecule that carries its specific amino acid
together.
Messenger RNA: mRNA carries genetic
information from the nuclear DNA to the
cytosol, where it is used as the template
for protein synthesis.
Special structural characteristics of
eukaryotic mRNA include a long sequence
of adenine nucleotides (a "poly-A tail") on
the 3'-end of the RNA chain, plus a'cap" on
the 5'-end .
RNA IS SYNTHESIZED FROM A DNA TEMPLATE BY AN RNA
POLYMERASE
The process of RNA synthesis is called transcription, and its substrates
are ribonucleoside triphosphates. The enzyme that synthesizes RNA is
RNA polymerase
The processes of DNA and RNA synthesis are similar in that
they involve:
(1)general steps of initiation, elongation, and termination with 5'
to 3'
(2) large, multicomponent initiation complexes.
(3)adherence to Watson–Crick base-pairing rules.
DNA and RNA synthesis do differ in several important ways,
including:
(1) ribonucleotides are used in RNA synthesis rather than
deoxyribonucleotides.
(2) U replaces T as the complementary base for A in RNA.
(3) only portions of the genome are vigorously transcribed or
copied into RNA, whereas the entire genome must be copied during
DNA replication
(4) there is no highly active, efficient proofreading function during
RNA transcription.
The strand that is transcribed or copied into an RNA molecule is referred
to as the template strand of the DNA.
the non-template strand, is frequently referred to as the coding strand
of that gene.The information in the template strand is read out in the 3'
to 5' direction.
Steps in RNA synthesis:
The process of transcription of a typical gene can be divided into three
phases:
initiation, elongation, and termination.
1. Initiation of transcription: involves the binding of the RNA
polymerase to a region on the DNA that determines the specificity of
transcription of that particular gene. That DNA sequence is known as
the promoter region .
many different promoters are recognized by prokaryotic RNA
polymerase ,include:
a.Pribnow box: This is a stretch of six nucleotides (5'-TATAAT-3')
b. -35 sequence: A second consensus nucleotide sequence (5-TTGACA3,), [A mutation in either the Pribnow box or the -3 5 sequence can
affect the transcription of the gene controlled by the mutant promoter.]
Elongation: the promoter region has been recognized by the
holoenzyme. RNA polymerase begins to synthesize a transcript of the
DNA sequence (usually beginning with a purine).
Unlike DNA polymerase, RNA polymerase does not require a primer and
has no known endonuclease or exonuclease activity. It therefore, has no
ability to repair mistakes in the RNA.
RNA polymerase uses ribonucleoside triphosphates, and releases
pyrophosphate each time a nucleotide is added to the growing chain.
Termination: The process of elongation of the RNA chain continues until
a termination signal is reached. termination requires an additional
protein :
P-Rho-independent termination , requires that the newly synthesized
RNA have two important structural features.
1. the RNA transcript must be able to form a stable hairpin turn .
2. palindrome .[A palindrome is a region of double-stranded DNA in
which each of the two strands have the same nucleotide sequence when
read in the same (for example, 5'->3' ) beyond the hairpin turn.
Some antibiotics prevent bacterial cell growth by inhibiting RNA
synthesis. For example, rifampin inhibits the initiation of
transcription by binding to the β-subunit of prokaryotic RNA
polymerase, thus interfering with the formation of the first
phosphodiester bond .Rifampin is useful in the treatment of
tuberculosis.
• D actinomycin :It binds to the DNA template and interferes with
the movement of RNA polymerase along the DNA.
TRANSCRIPTION OF EUKARYOTIC GENES
Promoters of eukaryotic genes:
1. TATA or Hogness box .
2. CAAT box .
3. GC box (GGGCGG).
Nuclear RNA polymerases of eukaryotic cells: There are three distinct
classes of RNA polymerase in the nucleus of eukaryotic cells:
1. RNA polymerase I : synthesizes the precursor of the large ribosomal
RNAs in the nucleolus.
2. RNA polymerase II: This enzyme synthesizes the precursors of
messenger RNAs also synthesizes certain small nuclear RNAs (snRNA
Inhibitors of RNA polymerase II: This enzyme is inhibited by αamanitin— potent toxin produced by the poisonous mushroom. Forms
a tight complex with the polymerase, thereby inhibiting mRNA synthesis
and, ultimately, protein synthesis.
3. RNA polymerase III : produces the small RNAs, including tRNAs, the
small 5S ribosomal RNA, and some snRNAs.
POSTTRANSCRIPTIONAL MODIFICATION OF RNA
Protein Synthesis
• The pathway of protein synthesis is called translation because the
"language" of the nucleotide sequence on the mRNA is translated into
the language of an amino acid sequence.
• The process of translation requires a genetic code, through which the
information contained in the nucleic acid sequence is expressed to
produce a specific sequence of amino acids.
• Codons are usually presented in the messenger RNA language of
adenine (A), guanine (G), cytosine (C), and uracil (U). Their nucleotide
sequences are always written from the 5'-end to the 3'-end. The four
nucleotide bases are used to produce the three-base codons. For
example, the codon 5-AUG-3' codes for methionine. Sixty-one of the 64
codons code for the twenty common amino acids.
Termination ("stop" or "nonsense") codons: Three of the codons,
UAG, UGA, and UAA, do not code for amino acids, but rather are
termination codons. When one of these codons appears in an mRNA
sequence, it signals that synthesis of the peptide chain is completed.
Characteristics of the genetic code include the following:
1. Specificity: The genetic codes specific that is, a specific
codon always codes for the same amino acid.
2. Universality: the specificity of the genetic code has been
conserved from very early stages of evolution.
3. Redundancy: Although each codon corresponds to a single
amino acid, a given amino acid may have more than one
triplet coding for it. For example, arginine is specified by six
different codons .
4. Nonoverlapping and commaless, that is, the code is read
from a fixed starting point as a continuous sequence of bases,
taken three at a time. For example ABCDEFGHIJKL , is read
as ABC/DEF/GHI/JKL without any "punctuation" between the
codons.
mutation :Changing a single nucleotide base on the mRNA chain (a
"point mutation") can lead to any one of three results
1. Silent mutation: The codon containing the changed base may code
for the same amino acid. For example, if the serine codon UCA is given a
different third base—U—to become UCU, it still codes for serine.
Therefore, this is termed a "silent" mutation.
2. Missense mutation: The codon containing the changed base may
code for a different amino acid. For example, if the serine codon UCA is
given a different first base—C—to become CCA, it will code for a
different amino acid, in this case, proline. This substitution of an
incorrect amino acid is called a "missense" mutation.
3. Nonsense mutation: The codon containing the changed base may
become a termination codon. For example, if the serine codon UCA is
given a different second become UAA, thenew codon causes termination
of translation at that point. The creation of a termination codon at an
inappropriate place is calleda "nonsense" mutation.
COMPONENTS REQUIRED FOR TRANSLATION
A large number of components are required for the synthesis of a
polypeptide chain. These include
1) all the amino acids that are found in the finished product.
2) the mRNA to be translated.
3) Transfer RNA: A specific type of tRNA is required per amino acid.
4) functional ribosome .
5) energy sources.
6) enzymes, as well as protein factors needed for initiation, elongation,
and termination of the polypeptide chain.
STEPS in PROTEIN SYNTHESIS
The pathway of protein synthesis translates the three-letter alphabet of
nucleotide sequences on mRNA into the twenty-letter alphabet of amino
acids that constitute proteins. The mRNA is translated from its 5'-end to
its 3'-end, producing a protein synthesized from its amino-terminal end
to its carboxyl-terminal end.
The process of translation is divided into three separate steps:
initiation, elongation, and termination.
A. Initiation The codon AUG at the beginning of the message is
recognized by a special initiator tRNA that enters the ribosomal P,
site. [ Only the initiator tRNA goes to the P site- other charged tRNAs
enter at the A site.
B. Elongation of the polypeptide chain involves the addition of amino
acids to the carboxyl end of the growing chain. During elongation, the
ribosome moves from the 5‘- end to the 3‘-end of the mRNA that is
being translated. After the peptide bond has been formed, the ribosome
advances three nucleotides toward the 3'-end of the mRNA. This causes
movement of the uncharged tRNA into the ribosomal E site (before
being released) and movement of the peptidyl-tRNAinto the P site
C. Termination occurs when one of the three termination codons moves
into the A site. These codons are recognized by release factors:
RF-1, which recognizes the termination codons UAA and UAG.
RF-2, which recognizes UGA and UAA.
RF-3,which binds GTP and stimulates the activity of RF-1 and RF-2.
These factors cause the newly synthesized protein to be released from
the ribosomal complex, and cause the dissociation of the ribosoma from
ricin (from castor beans) is a very potent toxin that exerts its effects'"by
removing an adenine from 28S ribosomal RNA, thus inhibiting
eukaryotic ribosome.