NUCLEIC ACIDS

Levels of Structure in Nucleic Acids

1. Primary structure: It is the order of bases in the polynucleotide sequence.
2. Secondary structure: It is the three-dimensional structure conformation of the backbone.
3. Tertiary structure: It is specifically the supercoiling of the molecule.
2 Principal Types of Nucleic Acids
 The differences between both nucleic acids can be seen in the secondary and tertiary structures.
 There is no direct analogy between the quaternary structures of protein and the interaction of nucleic acids
with other classes of macromolecules to form complexes, but it is similar to the interactions of the subunits in
an oligomeric protein.
1. DNA (deoxyribonucleic acid)
2. RNA (ribonucleic acid)
Covalent Structure of Polynucleotides
 Monomer: It is the smallest single unit of polymer.
 Nucleotides: These are the monomers of nucleic acids.
 One nucleotide consists of 3 parts which are covalently bonded to one another:
o Nitrogenous bases:
o Sugars:
o Phosphoric residue: Phosphates are normally joined to the C5 hydroxyl of the ribose or deoxyribose
sugar (designated 5’). Mono-, di-, and triphosphates are common. It makes the nucleotide negatively
charged.
 The order of bases in the nucleic acids of DNA contains the information necessary to produce the correct
amino acid sequence in the cell’s proteins.
Structures and Components of nucleotides
1. Nucleoside: It is a compound that consists of a base and a sugar covalently linked together. The base forms a
glycosidic linkage with the sugar. (Glycosidic bond links a sugar and other forms of moiety.)
- The sugar is linked to a nitrogen in both cases which can be called as an N-glycosidic bond. The glycosidic
linkage is from the C-1’ carbon of the sugar to the N-1 nitrogen of pyrimidines or to the N-9 nitrogen of purines.
a. Nucleic acid bases: These are also called nucleobases. It refers to a one- or two- ring nitrogenous
aromatic compound. Base is usually modified by methylation. The ring atoms of the base are
numbered.
i. Pyrimidine bases: It is a single-ring aromatic compound.
 1. Cytosine: It is found in both RNA and DNA.
2. Thymine: In DNA, it is substituted for Uracil. It is sometimes found in small extent in some forms of
RNA.
3. Uracil: It only occurs in RNA.
ii. Purine bases: These are double-ring aromatic compounds. These are both found in the DNA

and RNA.
1. Adenine:
2. Guanine:
b. Sugars: The ring atoms of the sugar are prime numbered.
i. β-D-ribose: The resulting compound will be a ribonucleoside.
ii. β-D-deoxyribose: The resulting compound will be a deoxyribonucleoside. It results from a lack
of oxygen

2. Nucleotide: It is formed when a phosphoric acid is esterified to

one of the hydroxyl groups of the sugar portion of a nucleoside
(usually at the 5th Carbon).
- It is named for the parent nucleoside, with the suffix -
monophosphate added. The position of the ester is specified by
the number of carbon atom at the hydroxyl group to which it is
esterified.
 - 5’ nucleotides are more commonly encountered. If there are more phosphate groups forming anhydride linkages
to the first phosphate attached, it will become a nucleoside diphosphate (2 phosphate groups), and a nucleoside
triphosphate (3 phosphate groups are attached).
Functions of Nucleotides
1. They carry chemical energy in their easily hydrolyzed phosphoanhydride bonds.
2. They combine with other groups to form coenzymes.
3. They are used as specific signaling molecules in the cell.
Forming Nucleic Acids
- The polymerization of nucleotides give rise to nucleic acids. The linkage between monomers in nucleic acids
involves formation of two ester bonds by phosphoric acid. The hydroxyl groups to which the phosphoric acid is
 esterified are those bonded to the 3’ and 5’ carbons on adjacent residues. This results to a 3’, 5’-phosphodiester
bond.
- Nucleotides are joined together by a phosphodiester linkage between 5’ and 3’ carbon atoms to form nucleic
acids. The linear sequence of nucleotides in a nucleic acid chain is commonly abbreviated by a one-letter code,
such as A-G-C-T-T-A-C-A with the 5’ end of the chain at the left.
- The nucleotide residues of nucleic acids are numbered from the 5’ end, which normally carries a phosphate
group, to the 3’ end, which normally has a free hydroxyl group.
- The sugar-phosphate backbone repeats itself down the length of the chain.
- The most important features of the structure of nucleic acids are the identities of the bases.
- In one system of notation:
o Single letters: It represents the individual bases like A, G, C, U, and T.
o Vertical lines: It shows the positions of the sugar moieties to which the individual bases are attached.
o Diagonal line through the letter P: It represents a phosphodiester bond.
- In a more common system of notation:
o Single letters: It is used to show the order of basses.
o Letter P:
 If written to the left of the single letter single-letter code for the base, it represents a a 5’ nucleotide.
 If it is written to the right, it represents a 3’ nucleotide.
 Example:
 pA: 5’-adenosine monophosphate (5’-AMP)
 Ap: 3’- adenosine monophosphate (3’-AMP)
 pGpApCpApU can be represented as GACAU, with the phosphates understood
The Structure of DNA
Double helix: It was proposed by James Watson and Francis Crick in 1953. A deoxyribonucleic acid (DNA)
molecule consists of two long polynucleotide chains composed of four types of nucleotide subunits. Each of these
chains is known as a DNA chain, or a
DNA strand.
The three-dimensional structure
of DNA – the DNA double helix –
arises from the chemical and structural
features of its two polynucleotide
chains.
 All the bases are on the inside
of the double helix
 The sugar-phosphate backbones
are on the outside.

- The X-ray diffraction pattern of

DNA demonstrated the helical
structure and the diameter.
o A complete turn of the helix
spans 10 base pairs,
covering a distance of 34Å
(3.4 nm).
  Individual base pairs: These are spaced 34Å (3.4 nm) apart.
 The places where the strands cross hide base pairs that extend perpendicular to the viewer.
 The inside diameter is 11Å (1.1 nm).
 The outside diameter is 20Å (2.0 nm).
 Within the cylindrical outline of the double helix are two grooves, which are empty spaces. Both are
large enough to accommodate polypeptide chains. Both can be sites at which drugs or polypeptides
bind to DNA.
 Minor groove: Small groove
 Major groove: Large groove
 The minus signs alongside the strands represent the many negatively charged phosphate groups along
the entire length of each strand.
 Phosphate: each group of the backbone carries a negative charge at neutral, or physiological pH.
 In order to neutralize the negative charges, positively charged ions, such as Na+ or Mg2+, and
polypeptides with positively charged side chains must be associated with DNA.
 In the eukaryotic DNA, it is complexed with histones in the cell nucleus.
- Chemical analyses showed that the amount of A was always the same amount of T, and the amount of G always
equaled the amount of C.
- Both of these lines of evidence were used to conclude that the DNA consists of to polynucleotide chains
wrapped around each other to form a helix.
- Hydrogen bonds between bases on opposite chains determine the alignment of the helix, with the paired bases
lying in planes perpendicular to the helix axis.
- The sugar-phosphate backbone is the outer part of the helix.
- The chains run in antiparallel directions, one in the 3’ to 5’ direction, while the other in the 5’ to 3’ direction.
- Complementary: With the combination of the results from the X-ray diffraction and chemical analyses, this kind
of base pairing means that adenine pairs with thymine while guanine pairs with cytosine.
o As the complementary base pairing occurs along the entire double helix, the two chains are also referred as
complementary strands.
o An adenine—thymine (A—T) base pair has two hydrogen bonds between the bases
o A guanine—cytosine (G—C) base pair has three hydrogen bonds.
o The distance between the points of attachment of the bases to the two strands of the sugar-phosphate
backbone is the same for the two base pairs (A—T and G—C), about 11Å (1.1 nm), which allows for a
double helix with a smooth backbone and no overt bulges.
A-DNA:
- Has 11 base pairs for each turn of the
helix
- Its base pairs are not perpendicular but
lie at an angle of about 20 degrees
- Right- handed helix
- It was originally found in DNA samples,
and was believed as an artifact of DNA
preparation.
- DNA: RNA hybrid can adopt an A-DNA
formation because the 2’-hydroxyl on
the ribose prevents a RNA helix from
adopting the B form.
- RNA: RNA hybrids may be found in the
A form.
B-DNA:
- It is considered as the normal, physiological form of DNA.
- It was predicted from the nature of the hydrogen bonds between purines and pyrimidines and later found
experimentally.
Z-DNA:
- Left-handed
- It is most often seen when there is a alternating sequence of purine-pyrimidine, such as dCpGpCpGpCpG
- It can play a role in the regulation of gene expression.
- It can be considered as derivative form of B-DNA, produced by flipping one side of he backbone 180 degrees
without having to break either the backbone or the hydrogen bonding of the complementary bases.
Base-stacking: It is the process in which the ring portions of the DNA bases are very hydrophobic and interact with
each other via hydrophobic bonding of their pi-cloud electrons.
- Even single-stranded DNA tends to form structures in which the bases can stack.
- In standard B-DNA, each base pair is rotated 32 degrees with respect to the preceding on. This form is perfect
for maximal base paring, but it is not optimal for maximal overlap of the bases. The edges of the bases that are
exposed to the minor groove must come in contact with water.in this form.
- Propeller-twist: The base-pairing distances are less optimal, but the base stacking is more optimal, and water is
eliminated from the minor-groove contacts with the bases.
- Besides twisting, bases also slide sideways, allowing them to interact better with the bases above and below
them. The twist and slide depends on which bases are present, it is actually a dinucleotide with its
complementary pairs. This is called a step in the nomenclature of the DNA structure.
Supercoils: These are the extra twists in the DNA structure..
Prokaryotes
1. Prokaryotic DNA is circular, and it forms supercoils.
2. DNA supercoiling is analogous twisting or untwisting a rope so that it is torsionally stressed.
3. Negative supercoils: This happens if strands are underwound.
a. It has a fewer number than the normal number of turns.
b. It introduces a torsional stress that favors unwinding of the right-handed B-DNA double helix.
4. Positive supercoils: These happen if the strands are overwound.
 a. It has more turns than the normal number of turns.
b. It overwinds a right-handed B-DNA double helix.
5. Naturally occurring circular DNA is negatively supercoiled except during replication, when it becomes
positively supercoiled. Cellular regulation of this process is critical.
6. Topoisomerases: Enzymes that are involved in changing the supercoiled state of DNA.
a. Class I: It cuts the phosphodiester backbone of one strand of DNA, pass the other end through,
and then reseal the backbone.
b. Class II: It cuts both strands of DNA, pass some of the remaining DNA helix between the cut
ends, and then reseal.
i. DNA gyrase is an example and it gives negative supercoils.
7. Ultracentrifugation: It can be used to detect supercoiled DNA because it sediments more rapidly than the
relaxed form.
Eukaryotes
1. Eukaryotic DNA is complexed with a number of proteins, especially with basic proteins that have
abundant positively charged side chains at physiological (neutral) pH.
2. Chromatin: It is the resulting material of an electrostatic attraction between the negatively charged
phosphate groups on the DNA and the positively charged groups on the proteins favors the formation
of complexes of this sort.
3. The topological changes induced by supercoiling must be accommodated by the histone-protein
component of chromatin.
4. Histones: These are the principal proteins in chromatin.
a. It has five main types : H1, H2A, H2B H3, an H4.
b. All these proteins contain large numbers of basic amino acid residues, such as lysine and
arginine.
c. DNA is tightly bonded to all except H1, for it is comparatively easy to remove from chromatin.
5. Chromatin resembles beads on a string, as it reflects the molecular composition of the protein-DNA
complex.
6. Each “bead” is a nucleosome, consisting of DNA wrapped around a histone core. This protein core
is an octamer, which includes two molecules of each type of histone but H1; the composition of the
octamer is (H2A)2(H2B)2(H3)2(H4)2
7. The “string” portions are called spacer regions; they consist of DNA complexed to some H1 histone
and nonhistone proteins. As the DNA coils around the histones in the nucleosome, about 150 base
pairs are in contact with the proteins; the spacer region is about 30 to 50 base pairs long.
8. Histones can be modified by acetylation, methylation, phosphorylation, and ubiquitinylation.
9. Ubiquitin is a protein involved in the degradation of other proteins.
Although enormously long strings of nucleosomes form on the chromosomal DNA, chromatin in a
living cell probably rarely adopts the extended “beads-on-a-string” form. Instead, the nucleosomes are
packed on top of one another, generating arrays in which the DNA is even more highly condensed.

DNA Denaturation
 - Melting: Heat denaturation of DNA. It can be monitored experimentally by observing the absorption of
ultraviolet light.
- Hyperchromicity: The bases absorb light in the 260-nm-wavelength region. As the DNA is heated and the
strands separate, the wavelength of absorption does not change, but the amount of light absorbed increases.
- It is based on the fact that the bases, which are stacked on top of one another in native DNA, become unstacked
as the DNA is denatured.
- The higher the percentage of G—C base pairs, the higher the melting temperature of a DNA molecule.
- The double helix unwinds when the DNA is denatured, with eventual separation of the strands. The double helix
is re-formed on renaturation with slow cooling and annealing.
- Renaturation of denatured DNA is possible on slow cooling. The separate strands can recombine and form the
same base pairs responsible for maintaining the double helix.

Principal Kinds of RNA and their Structures

- There are 6 kinds of RNA which play an important role in the life
processes of the cells:
1. Transfer RNA (tRNA)
a. It is a single-stranded polynucleotide chain,
between 73 and 94 nucleotide residues long, that
generally has a molecular mass of about 25,000 Da
(Dalton-atomic mass).
b. Smallest of three important RNAs.
c. It can be found in every living cell because at least
one tRNA bonds specifically to each of the amino
acids that commonly occur in proteins.
d. Cloverleaf structure: Duplexes in A-helical form
can be drawn as this and can be considered as
secondary structure of tRNA because it shows the
hydrogen bonding between certain bases.
e. Stems: The hydrogen-bonded portions of the
molecule.
f. Loops: The non-hydrogen bonded portion of the
molecule.
To produce a tertiary structure particularly necessary for tRNA to
interact with the enzyme that covalently attaches the amino acid to the
2’or 3’end, the tRNA folds into an L-shaped conformation that has been determined by X-ray diffraction. During
protein synthesis, both tRNA and mRNA are bound to the ribosome in a definite spatial arrangement that ultimately
ensures the correct order of the amino acids in the growing polypeptide chain.
2. Ribosomal RNA (rRNA)
a. Ribosomal RNA + proteins = site of protein synthesis
b. In both prokaryotes and eukaryotes, a ribosome consists of two subunits, one larger than the
other.
i. The smaller subunit consists of one large RNA molecule and about 20 different proteins
ii. The larger subunit consists of two RNA molecules in prokaryotes (3 in eukaryotes) and
about 35 different proteins in prokaryotes (about 50 in eukaryotes)
c. Analytical Ultracentrifugation: It is used for monitoring the dissociation and reassociation of
the ribosomes.
 i. Sedimentation coefficient: It characterizes the motion of the particle and it is expressed
in Svedberg units (S).
ii. The S value increases with the molecular weight of the sedimenting particle, but it is not
directly proportional to it because the particle’s shape also affects its sedimentation rate.
1. Prokaryotic ribosomes: has a sedimentation coefficient of 70S and when
dissociated:
a. Light 30S subunit containing a 16S rRNA and 21 different proteins
b. Heavy 50S subunit containing a 5S rRNA, 23s rRNA, and 34 different
proteins.
2. Eukaryotic ribosomes: has a sedimentation coefficient of 80 S and when
dissociated:
a. Small 40S subunit containing an 18S rRNA
b. Large subunit contains three types of rRNA molecule: 5S, 5.8S, and 28S.
3. Messenger RNA (mRNA)  directing protein synthesis
a. It is the least abundant of the main types of RNA.
b. The sequences of bases in mRNA specify the order of the amino acids in proteins.
c. It reflects the sequence of DNA bases in the gene that codes for that protein, although this
mRNA sequence is often altered after it is produced from the DNA.
d. It is logical that mRNA is formed when it is needed, directs the synthesis of proteins, and then
is degraded so that the nucleotides can be recycled.
e. It is usually the one that turns over most rapidly in the cell. Both tRNA and mRNA (as well as
ribosomes themselves) can be recycled intact for many rounds of protein synthesis.
f. In prokaryotes, there is no nuclear membrane, so mRNA can direct the synthesis of proteins
while it is still being transcribed.
g. Eukaryotic mRNA undergoes considerable processing. One of the most important parts of the
process is splicing out intervening sequences (introns), so that the parts of the mRNA that will
be expressed (exons) are contiguous to each other.
i. mRNA is initially formed as a larger precursor molecule called heterogenous nuclear
RNA (hnRNA)
ii. Intron: Lengthy intervening sequences that do not encode a protein. These are removed
by posttranscriptional splicing.
iii. Protective units called 5’-caps and 3’poly (A) tails are added before the mRNA is
complete.
4. Small nuclear RNA (snRNA)
a. It is only found in the nucleus of eukaryotic cells, and are distinct from other RNA types.
b. They are involved in processing of initial mRNA transcription products to mature form suitable
for export from the nucleus to the cytoplasm for translation.
c. In the cell, it is complexed with proteins forming small nuclear ribonucleoprotein particles
(snRNPs)
i. Its function is to help with the processing of the initial mRNA transcribed from DNA
into a mature form that is ready for export out of the nucleus.
5. Micro RNA (miRNA)
a. It has been found to be part of one of the oldest evolutionary relationships, that between
bacteria and bacteriophages.
b. Bacteria produce these, which then bind to sequences of the phage DNA, preventing their
infection.
c. It is also found to be important in repairing nerve damage in muscles.
6. Small interfering RNA (siRNA)
a. RNAi interference (RNAi): This is a process in which it was first discovered in pants and later
in mammals.
 b. It is also extensively by the scientists to eliminate the effect of a gene to help discover its
function. (siRNAs)
c. siRNAs are the main players in RNAi.
- It participates in the protein synthesis in a series of reactions ultimately directed by the base sequence of the
cell’s DNA.
- The base sequences of all types of RNA are determined by that of DNA.
- Transcription: This is the process by which the order of bases is passed from DNA to RNA.
- Ribosomes: These are the sites for assembly of the growing polypeptide chain in protein synthesis, in which
RNA is associated with proteins.
- Amino acids are brought to the assembly sit covalently bonded to tRNA, as aminoacyltRNAs.
- Translation: The process where the order of bases in mRNA specifies the order of amino acids in the growing
protein.
The Roles of Different Kinds of RNA
RNA type Size Function
Transfer RNA Small Transports amino acids to site of
protein synthesis
Ribosomal RNA Several kinds – variable in size Combines with proteins to form
ribosomes, the site of protein
synthesis
Messenger RNA Variable Directs amino acid sequence of
proteins
Small Nuclear RNA Small Processes initial mRNA to its
mature form in eukaryotes
Small interfering RNA Small Affects gene expression; used by
scientists to knock out a gene
being studied
Micro RNA Small Affects gene expression;
important in growth and
development

REPLICATION
The Flow of Genetic Information in the Cell
- The sequence of bases in DNA encodes genetic information.
- Replication: The duplication of DNA, giving rise to a new DNA molecule with the same base sequence as the
original, is necessary whenever a cell divides to produce daughter cells.
- The actual formation of gene products requires RNA
- Transcription: The production of RNA on a DNA template
- Three kinds of RNA are involved in the biosynthesis of proteins
o Messenger RNA is particularly important
- Translation: A sequence of three bases in mRNA specifies the identity of one amino acid in a manner directed
by the genetic code. The base sequence directs the amino acid sequence.
- The flow of genetic information is DNA  RNA  protein.
- Major exception are retroviruses in which they use RNA than DNA to direct the synthesis.
o RNA is their genetic material, but they also have a reverse transcriptase in which catalyzes the process.
Replication of DNA
- First step is to separate the two DNA strands
o The strands must be unwound to be separated from one another.
 o Nucleases: This preferentially attacks single-stranded DNA from which the cell must protect the unwound
portions from it.
- Second step is to synthesizing of DNA from the 5’to the 3’end
o Two antiparallel strands must be synthesized in the same direction on antiparallel templates
o The template has one 5’ 3’strand and one 3’ 5’ strand.
- Third step is to guard against replication errors
o It should be ensured that the correct base is added to the growing polynucleotide chain.
- Semiconservative Replication: This was performed by
Matthew Meselson and Franklin Stahl.
 o E. coli bacteria were grown with 15NH4Cl as the sole nitrogen source. In such a medium, all newly
formed nitrogen compounds, including purine and pyrimidine nucleobases, become labeled with 15N. The
15N-labeled cells were then transferred to a medium that contained only 14N. With every new generation
of growth, a sample of DNA was extracted and analyzed by density-gradient. DNA containing a 50–50
mixture of 14N and 15N appeared at a position halfway between the two bands after one generation, a
result to be expected with semiconservative replication.
- Direction of Replication
o Origin of Replication: A specific point in which the DNA double helix unwinds
o New polynucleotide chains are synthesized using each of the exposed strands as a template.
o Two possibilities exist for the growth of the new strands: synthesis can take place in both directions from
the origin of replication, or in one direction only.
o DNA synthesis is usually bidirectional.
o In each origin of replication, there are 3 points (replication forks) at which a polynucleotide chains are
formed.
o A “bubble” of newly synthesized DNA between regions of the original DNA is a manifestation of the
advance of the two replication forks in opposite directions.
o It is also called a θ structure because of its resemblance to the Greek letter theta.
o In prokaryotes: One such bubble and one origin of replication exists in the circular DNA.
o In eukaryotes: There are several origin of replications, and bubbles.
 The bubbles grow larger and eventually merge, giving rise to two complete daughter DNAs.
 Net chain growth: It represents the bidirectional growth of both new polynucleotide chains.
 Both new polynucleotide chain is synthesized in the 5’to 3’direction.
DNA Polymerase
- Semidiscontinuous DNA Replication
o All synthesis of nuclelotide chains occurs in the 5’  3’ direction from the perspective of the chain being
synthesized.
o Due to nucleophilic attacks, the hydroxyl group at 3’ attacks the incoming nucleotide 5’-triphosphate on its
sugar. Elimination of the pyrophosphate occurs while the formation of a phosphodiester bond happens.
o As the helix unwinds, the other parental strand (the 5’  3’ strand) is copied in a discontinuous fashion
through synthesis of a series of fragments 1000 to 2000 nucleotides in length, called the Okazaki
fragments; the strand constructed from Okazaki fragments is called the lagging strand.
o Because both strands are synthesized in concert by a dimeric DNA polymerase situated at the replication
fork, the 5’  3’ parental strand must wrap around in trombone fashion so that the unit of the dimeric
DNA polymerase replicating it can move along it in the 3’  5’ direction. This parental strand is copied in
a discontinuous fashion because the DNA polymerase must occasionally dissociate from this strand and
rejoin it further along. The Okazaki fragments are then covalently joined by DNA ligase to form an
uninterrupted DNA strand.
- DNA polymerase (E. Coli – Prokaryote): It catalyzes the successive addition of each new nucleotide to the
growing chain.
o DNA Polymerase I: It was
discovered first. It consists of a
single polypeptide chain .
o DNA Polymerase II & III: Multi-
subunit proteins that share some
common subunits.
 Polymerase II: It is strictly a
repair enzyme.
 o Pol IV and Pol V: These are both repair enzymes and are both involved in a unique repair mechanism
called the SOS response.
o Two important considerations regarding polymerases:
 Turnover Number: It is the speed of the synthetic reaction.
 Processivity: It is the number of nucleotides joined before the enzyme dissociates from the template.
o Polymerase III consists of a core enzyme responsible for the polymerization and 3’ exonuclease activity. It
contains α-subunits responsible for DNA binding, and y-complex which allows the β-subunits to form a
clamp that surrounds the DNA and slides along it as polymerization proceeds.
 DNA Polymerase III: It has the highest turnover and processivity.
o DNA polymerases cannot catalyze de novo synthesis. All three enzymes require the presence of a primer.
 Primer: It is a short oligonucleotide strand to which the growing polynucleotide chain is covalently
attached in the early stage of replication.
 In natural replication, RNA is the primer.
 DNA polymerases must have a nucleotide with a free 3’-hydroxyl already in place
so that they can add the first nucleotide as part of the growing chain.
o DNA polymerase reaction
 It requires all four deoxyribonucleoside triphosphates-- dTTP, dATP, dCTP, dGTP
 Mg2+ and a DNA template are also needed.
 It also needs all four ribonucleoside triphosphates – TTP, ATP, CTP, GTP
 These are all incorporated into the primer.
 Primer (RNA): It is hydrogen-bonded to the template (DNA).
 It provides a stable framework on which the nascent chain can start to grow.
 The newly synthesized DNA strand begins to grow by forming a covalent linkage to
the free 3’-hydroxyl group of the primer.
 DNA polymerase I: It has a specialized function in replication – repairing and patching DNA.
o The exonuclease activities are part of the proofreading-and-repair functions of DNA polymerases.
 It is a process by which incorrect nucleotides are removed from the polynucleotide so that the correct
nucleotides can be incorporated.
 The 3’  5’ exonuclease activity, which all three polymerases possess, is part of the proofreading
function.
 Incorrect nucleotides are removed in the course of replication and are replaced by the correct ones.
 The 5’  3’ exonuclease activity clears away short stretches of nucleotides during repair, usually
involving several nucleotides at a time.
 This is how RNA primers are removed.
Proteins Required for DNA Replication
Supercoiling and Replication
- DNA gyrase: It is an enzyme which catalyzes the conversion of relaxed, circular DNA with a nick I one strand
to the supercoiled form with the nick sealed that is found in normal prokaryotic DNA.
o It can sometimes cause a double-stranded break in DNA in the process of converting the relaxed, circular
form to the supercoiled form.
- A slight unwinding of the helix before the nick is sealed introduces the supercoiling.
- The energy required for the process is supplied by the hydrolysis of ATP.
Replication with Supercoiled DNA
- The prokaryotic DNA is negatively supercoiled in its natural state; however, opening the helix during
replication would introduce positive supercoils ahead of the replication fork.
- DNA gyrase fights these positive supercoils by putting negative supercoils ahead of the replication fork.
- Helicase: It is a helix-destabilizing protein which promotes unwinding by binding at the replication fork.
o DnaB Protein and rep protein

Single-stranded DNA protection for Replication

- Single-stranded regions of DNA are very susceptible to degradation by nucleases.
- Single-stranded binding protein (SSB) is another protein which stabilizes the single-stranded regions by binding
tightly to these regions of the molecule.
o The presence of this DNA-binding protein protects the single-stranded regions from hydrolysis by the
nucleases.
- The Primase Reaction
o RNA serves as primer in DNA replication.
o RNA can be formed de novo without a primer, while DNA requires a primer.
o A primer in DNA replication must have a free 3’-hydroxyl group to which the growing chain can attach,
and both RNA and DNA can provide this group.
o Primase: It is an enzyme in which is responsible for copying a short stretch of the DNA template strand to
produce the RNA primer sequence.
o The primer and the protein molecules at the replication fork constitute the primosome.
o The general features of DNA replication, including the use of an RNA primer, appear to be common in all
prokaryotes.
- Synthesis and Linking of New DNA Strands
o DNA polymerase III begins the synthesis of two new strands of DNA. The newly formed DNA is linked to
the 3’-hydorxyl of the RNA primer, and synthesis proceeds from the 5’ end to the 3’ end on both the
leading and the lagging strands.
o Two molecules of Pol III, one for the leading strand and the other for the lagging strand, are physically
linked to the primosome. It results to a protein complex called replisome.
o As the replication fork moves, the RNA primer is removed by polymerase I, using its exonuclease activity.
o The primer is replaced by deoxynucleotides, also by DNA polymerase I, using its polymerase activity.
(The removal of RNA primer and its replacement with the missing portions of the newly formed DNA
strand by polymerase I is the repair function)
o DNA ligase is the enzyme responsible for the final linking of the new strand.
Proofreading and Repair
- DNA replication occurs only once each generation in each cell.
- It is essential that the fidelity of the replication process be as high as possible to prevent mutations.
- Errors in replication occur spontaneously only one in every 109 to 1010 base pairs.
- Proofreading refers to the removal of incorrect nucleotides immediately after they are added to the growing
DNA during the replication process.
- During replication, a cut-and-patch process catalyzed by Polymerase I takes place. The cutting is removal of the
RNA primer by the 5’ exonuclease function of the polymerase, and the patching is the incorporation of the
required deoxynucleotides by the polymerase function of the same enzyme.
- Nick translation: It is a process in which DNA polymerase I is able to use its 5’  3’ exonuclease activity to
remove RNA primers or DNA mistakes as it moves along the DNA. It then fills it beind with its polymerase
activity.
- Mutagens: Agents that produce mutations like ultraviolet light, ionizing radiation, and various chemical agents
- In mismatch repair, enzymes recognize that two bases are incorrectly paired. The area with the mismatch is
removed, and DNA polymerases replicate the area again.
- Base-excision repair: A base that has been damaged by oxidation or chemical modification is removed by DNA
glycosylase, leaving an AP site, so called because it is apurinic or apyrimidinic (without purine or pyrimidine).
o An AP endonuclease then removes the sugar and phosphate from the nucleotide. An excision exonuclease
then removes several more bases. DNA polymerase I fills the gap, and DNA ligase seals the
phosphodiester backbone.
- Nucleotide-excision repair: It is common for DNA lesions caused by ultraviolet or chemical means, which often
lead to deformed DNA structures.

DNA Recombination
- Genetic Recombination: It is a natural process in which genetic information is rearranged to form new
associations.
o It is the exchange of one DNA sequence with another or the incorporation of a DNA sequence into another
o Homologous Recombination: If the combination involves a reaction between homologous sequences.
o Nonhomologous Recombination: It is the recombination of very different nucleotide sequences.
o Hot spots: Zones where a chromosome much more likely to show recombination.
Eukaryotic DNA Replication
- Replicators: Multiple origins of replication where eukaryotic chromosomes began to accomplish the DNA
synthesis
- Replicons: Zones where replication is proceeding
The Eukaryotic Cell Cycle
- M phase: Stage of Mitosis and cell division
- G1: means Gap; rapid growth and metabolic activity
- G0 : cells that are quiescent, not growing or dividing
- S phase: Time of DNA synthesis which is followed by the G2
- G2- relatively short period of growth in which the cell prepares for division
Replication and Cell Division
- Cells which have reached the G1 phase are competent to initiate the DNA replication.
- Proteins are involved in the control of replication and its link to the cell cycle.
- Origin replication Complex: It is a multi-subunit protein which initiates replication by binding to the origin of
replication; It is bound to the DNA throughout the cell cycle, but it serves as an attachment site for several
proteins that help control replication
- Replication Activator Protein: It is an activation factor which binds after the ORC
- Replication Licensing Factors: It binds after the activator protein is bound; replication cannot proceed until it is
bound
o Some RLFs are cytosolic – it will only have access to the chromosome once the nuclear membrane
dissolves during mitosis; replication will not occur until it is bound
o It makes the DNA competent after replication
- Pre-replication complex (pre-RC): It is the combination of the DNA, ORC, RAP, and RLFs
- Cyclins: Proteins that are produced in one part of a cell cycle and degraded in another.
- Cyclin-dependent protein kinases: These are protein kinases which cyclins are able to combine with.
o With the combination of the cyclins & CDKs, it activates DNA replication and also block assembly of a
pre-RC after initiation.
o Its state of activity determines the window opportunity for DNA synthesis.
o These complexes phosphorylate sites on the RAP, the RLFs, and the ORC itself.
o Once it has phosphorylated, the RAP dissociates from the pre-RC, as do the RLFs.
o Once phosphorylated and released, the RAP and the RLFs are degraded.
o Activation of the cyclin-CDKs serves both as an initiation of DNA replication and to prevent formation of
another pre-RC.
- In the G2 phase, the DNA has been replicated.
- During mitosis, the DNA is separated into daughter cells while the dissolved nuclear membrane allows entrance
of the licensing factors that are produced in the cytosol so that each daughter cell can initiate a new round of
replication.
Eukaryotic Polymerase Function
Polymerase α Has most subunits
Has ability to make primers
Lacks a 3’  5’ proofreading activity
Has low processivity
Polymerase δ Principal DNA polymerase in Eukaryotes
Interacts with PCNA (proliferating cell nuclear
antigen)
PCNA Counterpart of Pol III
Functions as sliding clamp
Trimer of 3 identical proteins that surround the
DNA
Polymerase ε Involved in leading strand replication
May replace polymerase δ in lagging strand
synthesis
Polymerase β Repair enzyme
Polymerase γ Carries out DNA replication in mitochondria

Telomerase Replication
o In replication of the lagging strand, short RNA primes are added and extended by DNA polymerase. When
the RNA primer at the 5’ end of each strand is removed, there is no nucleotide sequence to read in the next
round of DNA replication.
o The result is a gap (primer gap) at the 5’ end of each strand.
o Sequences at the 3’ end that cannot be copied by conventional DNA replication.
 o Synthesis of telomeric DNA by telomerase extends the 5’ end of DNA strands, allowing the strands to be
copied by normal DNA replication.
The Eukaryotic Replication Fork
o DNA replication is semiconservative.
o There is a leading strand with continuous synthesis in the 5’  3’ direction and a lagging strand with
discontinuous synthesis in the 5’  3’ direction.
o An RNA primer is formed by a specific enzyme in eukaryotic DNA replication (same with prokaryotes),
but in this case the primase activity is associated with Pol α . The formation of Okazaki fragments (150-
200 nucleotides long ) is initiated by Pol α.
o After the RNA primer is made and a few nucleotides are added by Pol α, the polymerase dissociates and is
replaced by Pol δ and its attached PCNA protein.
o Replication Factor C is another protein which is involved in attaching PCNA to Pol δ.
o The RNA primer is degraded, but the polymerase does not have the 5’  3’ exonuclease activity to do it.
o Separate enzymes, FEN-1 and RNase H1, degrade the RNA.
o Continued movement of Pol δ fills in the gaps made by primer removal.
- For prokaryotic replication, topoisomerases relieve the torsional strain from unwinding the helix, and a single-
stranded binding protein, called RPA, protects the DNA from degradation.
- Finally, DNA ligase seals the nicks that separate the fragments.
- In eukaryotic replication, histones are associated with DNA as it is formed. Histone biosynthesis occurs at the
same time and at the same rate as DNA biosynthesis.
 TRANSCRIPTION
Transcription: The process of making RNA from DNA and it is the major control point in the expression of genes and
production of proteins.
- General Features of RNA Synthesis
o RNA is initially synthesized using a DNA template in the process called transcription
o DNA-dependent RNA polymerase: it is the enzyme that catalyzes the process.
o All four ribonucleoside triphosphates (ATP, CTP, GTP, UTP) are required, as is Mg2+
o A primer is not needed in RNA synthesis, but a DNA template is required
o As is the case with DNA biosynthesis, the RNA chain grows from the 5’  3’ end. The nucleotide at the
5’ end of the chain retains its triphosphate group (abbreviated ppp).
o The enzyme uses one strand of the DNA as the template for RNA synthesis. The base sequence of the
DNA contains signals for initiation and termination of RNA synthesis. The enzyme binds to the template
strand and moves along it in the 3’ to 5’ direction.
o The template is unchanged.
- Transcription in Prokaryotes
o RNA polymerase: It is the enzyme that synthesizes RNA.
 The actual composition of the
enzyme is α2ωββ’σ.
 The σ-subunit is loosely bound to the
core enzyme.
 Core enzyme: α2ωββ’
 Holoenzyme: It consists of al the
subunits, including the σ-subunit.
 σ-subunit: recognition of specific
promoter
 core enzyme: make an active site for
polymerization
o DNA strands used for transcription
 Template strand: It is the strand that directs the synthesis of the RNA.
 Antisense: Its code is the complement of the RNA that is produced.
 (-) strand by convention
 Coding strand: Its sequence of DNA will be the same as the RNA sequence that is produced.
 Sense strand: the RNA sequence is the sequence that we use to determine what
amino acids are produced in the case of mRNA.
 (+) by convention or even the nontemplate strand
 Core enzyme: Catalytically active but lacks specificity
 Holoenzyme of RNA: binds to specific DNA sequences and transcribes only the correct strand
 σ-subunit: recognition of promoter locus which is a DNA sequence that signals the start of RNA
transcription
 it is released after transcription begins and about 10 nucleotides have been added to
the RNNA chain
 Prokaryotes can have more than one type of σ-subunit.
 o Its nature can direct RNA polymerases to different promoters and cause the
transcription of various genes to reflect different metabolic conditions.
o Knowing where to begin transcription
 Promoters: These are DNA sequences that provide direction for RNA polymerase.
 The promoter region to which RNA polymerase binds is closer to the 3’end of the template strand
than is the actual gene for the RNA to be synthesized.
 The RNA is formed form the 5’end to the 3’end so the polymerase moves along the template strand
from the 3’end to the 5’end.
 The binding site for the polymerase is said to lie upstream of the start of transcription, which is farther
to the 5’side of the coding strand.
 The promoter sequence will be given based to the coding strand, even though the RNA polymerase is
actually binding to the template strand.
 Promoters are upstream, which means the 5’side of the coding strand and to the 3’side of the template
strand.
  The first base to be incorporated into the RNA chain is said to be at position +1 and is called the
transcription start site (TSS). All the nucleotides upstream from this start site are given negative
numbers.
 Pribnow box: It is the -10 region and the first promoter element is about 10 bases upstream.
 - 35 region or -35 element: Position where the next promoter element is 35 bases upstream of the TSS.
 Element: It is a general term for a DNA sequence that is somehow important in controlling
transcription.
 Core promoter: It is the area from the -35 element to the TSS.
 UP element: It can be located upstream of the promoter which enhances the binding of RNA
polymerase.
 Extended promoter: It is the region from the end of the UP element to the transcription start site.
 Consensus sequences: The base sequence of promoter regions has been determined for a number of
prokaryotic genes, and a striking feature is that they contain may bases in common.
 Promoter regions are A-T rich, with two hydrogen bonds per base pair for they are also easily
unwound.
o Chain initiation
 It is the first phase of transcription. It is also the part that is most controlled.
 It begins when RNA polymerase binds to the promoter and forms what is called the closed complex.
 The σ-subunit directs the polymerase to the promoter. It bridges the -10 and -35
regions of the promoter to the RNA polymerase core via a flexible “flap” in the σ-
subunit. Core enzymes lacking the σ-subunit bind to areas of DNA that lack
promoters. The holoenzyme may bind to “promoterless” DNA, but it dissociates
without transcribing.
 It requires formation of the
open complex.
 Portion of
β’ and the
σ-subunits
initiate
strand
separation,
melting
about 14
base pairs

surrounding the transcription start site.

 A purine ribonucleoside triphosphate is the first base in RNA, and it binds to its
complementary DNA base at position +1.
o Chain elongation
 After the strands have separated, a transcription bubble of about 17 base pairs moves down the DNA
sequence to be transcribed, and RNA polymerase catalyzes the formation of the phosphodiester bonds
between the incorporated ribonucleotides. When about 10 nucleotides have been incorporated, the s-
subunit dissociates and is later recycled to bind to another RNA polymerase core enzyme.
 The transcription process supercoils DNA, with negative supercoiling upstream of the transcription bubble and
positive supercoiling downstream. Topoisomerases relax the supercoils in front of and behind the advancing
transcription bubble. The rate of chain elongation is not constant. The RNA polymerase moves quickly through
some DNA regions and slowly through others. It may pause for as long as one minute before continuing.
 Abortive transcription: RNA polymerases releases most chains near the beginning of the process. Its cause is
the failure of RNA olymerase to break its own bonds to the promoter via the σ-subunit.
 In order for the elongation to occur, the RNA polymerase must be able to launch itself off the
promoter.
 Given the tight bonding between the σ-subunit and the promoter, this requires substantial energy.
 The RNA polymerase is bound
tightly to the DNA promoter. It
“scrunches” the DNA into
itself, causing torsional strain
of the separated DNA strands.
This provides the energy to
allow the polymerase to break
free.

o Chain Termination
 It involves specific sequences
downstream of the actual gene
for the RNA to be transcribed.
 Two types of termination
mechanisms:
 Intrinsic
termination
o It is

controlled by specific sequences called termination sites.

o Termination sites are characterized by two inverted repeats spaced by a few
other bases.
o Inverted repeats are sequences of bases that are complementary, such that
they can loop back on themselves.
o The DNA encodes a series of uracils.
 o When the RNA is created, the inverted repeats form a hairpin loop to stall
the advancement of RNA polymerase.
o The presence of the uracils causes a series of A-U base pairs between the
template strand and the RNA. A-U pairs are weakly hydrogen-bonded
compared with G-C pairs, and the RNA dissociates from the transcription
bubble, ending transcripton.
 Rho-dependent termination ρ
o It causes a hairpin loop to form.
o The ρ protein binds to the RNA and chases the polymerase.
o When the polymerase transcribes the RNA that forms a hairpin loop, it stalls,
giving the ρ protein a chance to catch up.
o When the ρ protein reaches the termination site, it facilitates the dissociation
of the transcription machinery. The movement of the ρ protein and the
dissociation require ATP.

Transcription Regulation in Prokaryotes

- In prokaryotes, the control of transcription is largely responsible for controlling the level of protein production.
- Transcription is controlled in four principal ways:
o Alternative σ factors
  Viruses and bacteria can exert some control over which genes are expressed by producing different σ-
subunits that direct the RNA polymerase to different genes. The virus has a set of genes called the early

genes, which are transcribed the host’s RNA polymerase, using its regular σ-subunit.
 One of the viral early gens codes for a protein called gp28.
 gp28: it is another σ-subunit which directs the RNA polymerase to transcribe preferentially more of
the viral genes during the middle phase.
 Products of the middle phase transcription are gp33 and gp34, which together make up another σ
factor that directs the transcription of the late genes.
 Remember that σ factors are recycled. As more and more of the gp28 is produced, it competes for
binding with standard σ for the RNA polymerase, eventually subverting the transcription machinery
for the virus instead of the bacterium.
o Enhancers
 The genes for ribosomal RNA production have three upstream sites, called Fis sites because they are
binding sites for the protein called Fis.
 These sites extend from the end of the UP element at -60 to -150, and are examples of a class of DNA
sequences called enhancers.
 Enhancers are sequences that can be bound by proteins called transcription factors.
 Difference between an enhancer and a promoter
 Promoter: When a DNA sequence is labeled, it implies that the RNA polymerase
binds to that region of DNA.
 Enhancer: It is a DNA sequence that is usually upstream of the promoter. The
polymerase does not bind to enhancers.
 o When enhancers allow a response to changing metabolic conditions, they are
usually referred to as response elements.
o When binding the transcription factor increases the level of transcription, the
element is said to be an enhancer.
o When binding the transcription factor decreases transcription, the element is
said to be a silencer.
o The position and orientation of enhancers is less important than for
sequences that are part of the promoter.
o Operons
 In prokaryotes, genes that encode enzymes of certain metabolic pathways are often controlled as
group, with the genes encoding the proteins of the pathway being close together and under the control
of a common promoter.
 Usually these genes are not transcribed all the time.
 Inducer: It is a suitable substance whose presence can be trigger the production of these proteins like
enzyme B-galactosidase in E. coli.
- Transcription in Eukaryotes
o Prokaryotes have a single RNA polymerase that is responsible for the synthesis of all three ids of
prokaryotic RNA -mRNA, tRNA, and rRNA. The polymerase can switch σ factors to interact with
different promoters, but the core polymerase stays the same.
o In eukaryotes, three primary RNA polymerases with different activities are known to exist.
o Each one transcribes a different set of genes and recognizes a different set of promoters:
 RNA polymerase I is found in the nucleolus and synthesizes precursors of most, but not all ribosomal
RNAs.
 RNA polymerase II is found in the nucleoplasm and synthesizes mRNA precursors.
 RNA polymerase III is found in the nucleoplasm and synthesizes the tRNAs, precursors of 5S
ribosomal RNA, and a variety of other small RNA molecules involved in mRNA processing and
protein transport.
o All three have two larger subunits that share sequence homology with the B- and B’- subunits of
prokaryotic RNA polymerase that make up the catalytic unit.
o There are no σ -subunits to direct polymerases to promoters,
o The detection of a gene to be transcribed is accomplished in a different way, and the presence of
transcription factors plays a larger role.
o Structure of RNA polymerase II
 C-terminal domain (CTD): It is found in the C-terminal region of the protein.
 Threonine, serine, and tyrosine are all substrates for phosphorylation, which is
important in the control of transcription initiation.
o Recognizing Right DNA to transcribe
 Pol II Promoters
 Upstream elements: It acts as enhancers and silencers.
o Enhancers: Specific binding proteins activate transcription above basal
levels
o Silencers: Specific binding proteins suppress transcription
o GC box and CAAT box: two common elements close to the core promoter
 TATA box: It is fount at position -25, which has a consensus sequence of TATAA
(T/A)
 Transcription start site at position +1 surrounded by a sequence called initiator
element. This sequence is not well conserved.
 Possible downstream regulator although more rare than upstream regulators.
 It is natural to lack one of the elements.
 The initiator plus the TATA box make up the core promoter and are the two most consistent parts
across different species and genes.
 “TATA-less” promoters: Genes do not have TATA boxes
 In some genes, the TATA box is necessary for transcription, and deletion of the TATA box causes a
loss of transcription.
 TATA boxes can also orient RNA polymerase correctly in others.
 Elimination of TATA boxes in these genes causes transcription at random starting points.
o Initiation of Transcription
 Transcription factor: It is any protein that regulates transcription but is not itself a subunit of RNA
polymerase.
 Transcription initiation begins by the formation of a preinitiation complex, and most of the control of
transcription occurs at this step.
 This complex normally contains RNA polymerase II and six general transcription factors (GTFs) –
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
o The general
transcription factors
are required for all
promoters.
o The first step in the
formation of the
preinitiation complex
is the recognition of
the TATA box by
TFIID.
o The transcription
factor is actually a
combination of
several proteins.
 o TATA-binding protein (TBP): it is the primary protein. It is a universal transcription factor and is highly
conserved.
 The TBP protein binds to the minor groove of the DNA at the TATA box via the least 180 amino
acids of its C-terminal domain.
 Once TFIID is bound, TFIIA binds, and RFIIA also interacts with both the DNA andTFIID. TFIIB
also binds to TFIID, bridging the TBP and Pol II. TFIIA and TFIIB can bind in either order, and they
do not interact each other. TFIIB is critical for the assembly for the assembly of the initiation complex
and for the location of the correct transcription start site. TFIIF then binds tightly to Pol II and
suppresses nonspecific binding. Pol II and TFIIF then bind stably to the promoter. TFIIF interacts
with Pol II, TBP, TFIIB, and the TAFIIs. It also regulates the activity of the CTD phosphatase.
 The last two factors to be added are TFIIE and TFIIH. TFIIE interacts with unphosphorylated Pol II.
These two factors have been implicated in the phosphorylation of polymerase II. TFIIH also has
helicase activity. After all these GTFs have bound to unphosphorylated Pol II, the preinitiation
complex is complete. TFIIH has been found to have other functions as well, such as DNA repair.
o Before transcription can begin, the preinitiation complex must form the open complex. In the open
complex, the Pol II CTD is phosphorylated, and the DNA strands are separated.
- Elongation and Termination
o Uses topoisomerase to have no surface tension.
o Pol II does not elongate efficiently when alone in vitro, and the difference lies in the elongation factors.
 TFIIF has a role in the formation of the preinitiation complex, and has stimulatory effect on
elongation.
 TFIIS
o It is controlled in many ways.
 Pause sites: Sequences where the RNA polymerase hesitates.
 It can also be aborted, leading to premature termination. Abortive termination happens more often
than correct elongation, usually just after few nucleotides have been linked.
 Antitermination: Elongation can proceed past the normal termination point.
 TFIIF class of elongation factors promotes a rapid read-through of pause sites, perhaps locking the
Pol II into an elongation-competent form that does not pause and dissociate.
 Arrest Release Factors: The TFIIS class of elongation factors which help the RNA polymerase move
again after it has paused.
 A third class of elongation factors consists of P-TEF and N-TEF proteins (positive-transcription
elongation factor and negative-transcription elongation factor). They increase the productive form of
transcription and decrease the abortive form, or vice versa.
 At some point during either elongation or termination, TFIIIF dissociates from Pol II.
o Termination begins by stopping the polymerase.
o AAUAAA: It is the eukaryotic consensus sequence for termination. This sequence may be 100-1000 bases
away from the actual end of the mRNA.
o After termination occurs, the transcript is released, and the Pol II open form (phosphorylated) is released
from the DNA. The phosphates are removed by phosphatases, and the Pol II / TFIIF complex is recycled
for another round of transcription.
- Transcription Regulation in Eukaryotes
o Basal level: low level of transcription in which only involves RNA polymerase and general transcription
factors to initiate transcription of mRNA.
o The actual transcription level of some genes may be many times the basal level.
o Activators: Gene-specific transcription factors which differs one from the other.
o Enhancers and Silencers: Regulatory sequences that augment or diminish transcription. Not all genes have
these sequences.
- Response Elements:
 o Response elements: Enhancers that respond to certain metabolic factors.
 These all bind proteins that are produced under certain cell conditions, and several related genes are
activated. It differs from an operon because the genes are not linked with one another and not under a
common promoter.
Posttranscriptional RNA Modification
- Three principal kinds of RNA (tRNA, rRNA, and mRNA) are all modified enzymatically after transcription to
give rise to the functional form of the RNA in question. The initial size of the RNA transcripts is greater than
the final size because of leader sequences at the 5’ end and trailer sequences at the 3’ end. The leader and trailer
sequences must be removed, and other forms of trimming are also possible. Terminal sequences can be added
after transcription, and base modification is frequently observed, especially in tRNA.
- Messenger RNA:
o In the C-terminal domain, Extensive processing takes place in eukaryotic mRNA. Modifications include
capping of the 5’end, polyadenylating (adding a poly-A sequence to) the 3’ end, and splicing of coding
sequences.
o These kinds of processing is not a feature of the synthesis of prokaryotic mRNA.
- 5’capping
o Phosphatase reaction: from 3 phosphates  2 phosphates
o Guanyltransferase: 2 phosphates will be removed but replaced by GTP
o Methyltransferase: methyl group is added to base
- mRNA modification
o The polyadenylate tail at the 3’ end of a message (typically 100 to 200 nucleotides long) is added before
the mRNA leaves the nucleus.
o It is thought that the presence of the tail protects the mRNA from nucleases and phosphatases, which
would degrade it.
o It also protects the mRNA from exonuclease degradation.
o The adenylate residues would be cleaved off before the portion of the molecule that contains the actual
message is attacked.
- Splicing
o Enzyme-assisted: It is enzyme assisted since it happens in the c-terminal domain to create lariat. Main
reaction in eukaryotes. It happens to the mRNA until it goes out of nucleus.
o Ribozymes: It can splice itself. End result is not lariat, but circular.
o The genes of prokaryotes are continuous; every base pair in a continuous prokaryotic gene is reflected in
the base sequence of mRNA.
o The genes of eukaryotes are not necessarily continuous, eukaryotic genes frequently contain intervening
sequences that do not appear in the final base sequence of the mRNA for that gene product.
o In splicing, small nuclear RNP(enzymes) helps in mRNA splicing, in the formation of the lariat. snRNA as
well as protein component is also present in the snRNPs (can also be ribozyme since it has RNA
component which helps access enzyme.)
o Called U because there are many U in sequence.
o Lariat will be taken off (introns). Then exons will be attached together. It leaves an exon junction complex
protein at the site of junction, it is a portion of mRNA.
o If there are no U, there may be exon skipping for there is no distinct ends indicated that should meet.

o Exons: The DNA sequences that are expressed (the ones actually retained in the final mRNA product)
o Introns: The intervening sequences which are not expressed; junk DNA not needed in making protein
o The expression of a eukaryotic gene involves not just its transcription but also the processing of the
primary transcript into its final form.
o When the gene is transcribed, the mRNA transcript contains regions at the 5’ and 3’ ends that are not
translated and several introns shown in green.
 o The introns are removed, linking the exons together. The 3’ end is modified by adding a poly-A tail and a
7-mG cap to yield the mature mRNA.

- Usually the last part, it happens in the C-terminal DNA. series of AAAAAA will be added.
- Polyadenylation protein will add the series of AAAAAA.
- To protect the 3; end from nucleases as it is single stranded mRNAs. Serves as a buffer as the endonucleases
will only attack the AAAs.
 - Alternative Splicing:
- It also happens in
the C-terminal
domain.
- Exon skipping is
not bad in
alternative splicing
because a
functional protein
is formed.
- Bad is when these
get dysfunctional,
one will get sick.
Usually happens in
splicing.

Introns Spliced out to

make Mature RNA
- Ribonucleoprotein particles in which are formed through association with a set of nuclear proteins and the
removal of intervening sequences which take place in the nucleus.
- The substrate for the splicing is the capped, polyadenylated pre-mRNA.
- Splicing requires cleavage at the 5’ and 3’ end of introns and joining of the two ends
- It has to leave the nuclear envelope through the nuclear pore.
- It has to pass through the nonsense mediated decay as a final checking of the mRNA if the protein is functional
or not to proceed to translation. If it does not pass, mRNA will eventually degrade.
Noncoding RNA: no protein will be produced
o Eukaryotes is made by RNA pol III
o RNA pol I makes rRNA sequences
- tRNA
- snRNA
- srRNA
- rRNA

Importance of Nucleolus: creation of ribosomes; where non coding RNAs or rRNAs create, where ribosomes are also
packaged and released to the part where it will be translated

TRANSLATION
- The ribosome is the site for protein synthesis. The mRNA and tRNA, which are bound to the ribosome in the
course of protein synthesis, are responsible for the correct order of amino acids in the growing protein chain.
- From nucleic acids  amino acids  proteins
- Genetic code: It reads triplet (ribosomes reads mRNA as three  codon), nonoverlapping (will not repeat),
commaless (no space in between), degenerate (many triplets can code for particular amino acids), universal code
(used by all organisms), which mRNA is read from 5’ to 3’
 o Start codon: AUG, Methionine
o Stop codon: UGA, UAA, UAG
o Wobble base: 3rd base from tRNA (5’position)
- Usually the first two nucleic acids are the same, they only differ at the
end.
- There are times where the base pairs with other bases.
- One particular nucleic acid called Inosine, its base is hypoxanthine,
almost the same as guanine and adenine. It is deaminated adenine or
guanine. It can H-bond to an A or a U.
- Goal of the ribosomes is to look for Methionine AUG as the start to read
the frames.

1. Amino acid activation

a. It involves both tRNA and a specific enzyme called
aminoacyl-tRNA synthetases.
b. 5’ 3’ mRNA; codon
c. 3’ 5’ tRNA complementary of codon; anti codon- has amino acid
d. ATP bonds first to tRNAS to be activated, it will be easier for tRNA to react with the amino
acid. It bonds in the carboxylic acid group. It binds in the 3’prime hydroxyl group of the ribose
of tRNA.
e. It becomes the activated amino acid, can start translation.
f. No elongation if no prior activation of tRNA happens because no amino acid will be passed
while reading the ribosome.
 g. It is catalyzed by tRNA synthetase.

Centrifugation unit  S (Svedberg) ; higher number  it will be the first one to fall
- It is usually composed of small subunit and large subunit
- 70S  50 S + 30S
- 80S  60S + 40S
- To differentiate bacterial to eukaryotic ribosome, identify the 16S rRNA.
1. Initiation
a. Small subunit binds to the 5’prime of the leftmost part of the RNA, together with the first
tRNA.
b. For prokaryotes, methionine is in a formyl group. Usually has 1 carbon and has acid. To
identify where to start, the first methionine is usually tagged with a formyl group. Next
methionine along the way is no longer formylated.
c. 30S and tRNA both looks for the Shine-Dalgarno sequence which is purine-rich(Gs and As)
where AUG can be seen.
d. Initiation factors
e. Both large and small subunits of the ribosome should be present in order for it to be functional.
f. First tRNA will be placed in the P-site.
g. Looks for a sequence before start codon.
2. Asa
a. Exit site:
b. Peptidyl site: where peptides lengthen
c. Aminoacyl site: when newly activated tRNA enters and binds
d. Ribosome has to move in order to read the next three triplet.
e. Translocation: First to move is large subunit. What used to be the P site will become an E site.
Once the large subunits move, the small subunit move.
3. Elongation
a. EF-Tu (elongation factor) brings the newly charged tRNA to the A site. If wrong, it will just be
released. This will react to the adjacent amino acid.
b. Elongation factor G: It is important in the translocation. It pushes the tRNAs to the P site and E
site.
i. 23s rRNA which is found in the Large Subunit participates in the reaction
ii. Ribosomal RNA: participates in the reaction making it a Ribozyme
4. Termination
a. It should no longer elongate so the Release factors occupy the A site so no tRNA will occupy
(with the aid of stop codons).
b. When the RF enters, the water will cleave the bond between the peptide and tRNA
c. Polysomes read the mRNA and produce protein
d. Since prokaryotes have no introns, magkakasunod ang genes nila, each gene has their own
Shine- Dalgarno Sequence ,so if 3 ribosomes bind too the mRNA, it will be read thrice
 M2+: if may nucleic acids it needs this metal to undergo metal ion catalysis is done
by the enzymes
Eukaryotic Translation

- Before translation, it has to undergo 5’ capping, splicing, and polyadenylation.

1. Initiation
a. The ribosome will bind to the 5’ end, left most of mRNA using the Methionine and eIFs
(eukaryote initiation factors)
i. eIFs bring the tRNAs to the P site
ii. small subunit will position itself at the 5’ end
iii. ribosome will scan for the AUG codon to mark the start of translation, no need for
shine-Dalgarno sequence
1. look for the first AUG, and this is where the large subunit will bind to
iv. Charged tRNA will enter at the A site
2. Elongation
 a. Almost same as prokaryotic elongation, only have different names of proteins involved
b. eEF1A as the eukaryotic counterpart of the EF-Tu
c. eEF2 as the eukaryotic counterpart of the EF-G
3. Translation Termination
a. There is only one release factor in eukaryotes.
b. Same mechanism as prokaryotes, when stop codon is scanned, release factors will be at the A
site, hydrolysis happens
c. Selenocysteine: translation does not stop even when it has encountered stop codon
d. Polysomes are also present in eukaryotes
4. Protein Folding
a. Proteins can fold on its own
b. Cells release heat-shock proteins to correctly fold protein if it was incorrectly folded
c. “like come here bb imma fix you”
d. If it did not degrade, it will accumulate and aggregate then cell will get broken
e. If protein has ubiquitin, it is already marked for degradation.
 ISOLATION AND MANIPULATION METHODS
- Amplifying of gene of interest in the laboratory
o In vivo: multiplying the gene of interest in another cell
 Bacteria are faster to divide so will yield more gene of interest than eukaryotic cells
 Vector: It is usually a plasmid (circular DNA which is in bacteria usually which replicates on its own,
has own Origin of replication) in which the gene of interest is cut and joined with the vector; once
dumami ang bacteria, dadami ang plasmid, dadami ang gene of interest, isolate the plasmid to get the
gene of interest
 Restriction endonucleases: to cut the DNA (restriction enzymes)
 Recognition sequence: if this is seen it is a mark of where it is to be cut
 Genetic recombination: combining of two different genes
 Recombinant DNA molecule: It is to be inserted to bacteria
 Proliferates more longer  more preferred
o In vitro amplification (polymerase chain reaction PCR): Multiplication in test tubes
 Usually in one reaction it will yield a lot
 Isolate the DNA, subject this particular DNA to a polymerase chain reaction (in vitro replication)
 Isolate the DNA containing the gene of interest
 Put into test tube and add deoxynucleotidetriphosphate (ATP, GTP, CTP, TTP)
  Add short nucleotide complementary to the gene of interest (DNA primer) bond
through hydrogen bonding
 DNA polymerase: only enzyme to be used
 Forward or Reverse Primer
 Add Mg2+ (as DNA polymerase works with metal ion catalysis)
 Denaturation: Splits into two without the helicase, temp of 95-100 degrees Celsius destroying
hydrogen bonds of double stranded helix
 Annealing: Bonding of primer and DNA
 Extension: Where DNA polymerase works, extends gene of interest only, does not replicate whole
DNA but only gene of interest
 It has to be repeated 30 times (denaturation, annealing, extension)
 Thermocycler 98-62-72 : equipment used in PCR
 After 30 times, the final extension
 Checks how DNA polymerase is strong to be used as it denatures in high temperatures
 TAQ polymerase coming from thermophilic bacteria is used
 Heat stable polymerase
 2n = equation for the number of genes (n=number of cycles)
 Goal of the PCR: since one gene is being amplified, only one band will be seen
- Analysis of Gene of Interests
o Agarose Gel Electrophoresis
 A gel where DNA extracts are run under electricity
 Negative end at taas (general charge of DNA are negative due to the phosphates)
 Positive end sa baba
 Direction of DNA is from top to bottom
 Bottom parts are smaller, larger parts are on the top
 Nearer to the top, slower travel
 M- Marker or Ladder: one will know the particular size
Restriction Fragment Length
Polymorphism (RFLP)
- Restriction enzyme will be
used
- There will be fragments of
different lengths
- Used to determine single
nucleotide mutations
- Used to diagnose some
diseases and
paternity/maternity testing

Blotting Methods:
- Final result will be gene of
interest only
- It is a yes or no , presence of blot  presence of DNA, RNA, Protein
DNA: Southern Blotting; to determine
if DNA is present or not
RNA: Northern Blotting
Protein: Western Blotting

Sanger Sequencing or Dideoxy

-addition of normal nucleotide
triphosphate but in sugar component, it
has no OH
- It will not form a
phosphodiester bond with next
incoming dNTP
- An OH is needed to form
phosphate-sugar backbone
- Without the H , replication will
be terminated
- Usual PCR will occur
OH is needed
 - 5’ end is at the bottom, 3’ end at the top (inferred sequence from the gel)
- Gene of interest: complement of PCR (5’ end at top, 3’ end at bottom)
New Generations Sequencing (NGS)
- It does not need gel
- But it uses colors
- dNTPS are labeled in fluorescent compounds
Genetic Manipulation
- Genetic recombination
- The bacteria is manipulated to express a gene
(trans gene) not even his
- Pros and Cons
- Food security

Genetic manipulation in Plants

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