Molecular biology

Hershey
and Chase
experiment
 Hershey and Chase experiment
 In the mid-twentieth century, scientists were still unsure as to whether DNA or protein was the genetic material of the
cell.
 It was known that some viruses consisted solely of DNA and a protein coat and could transfer their genetic material
into hosts.
 In 1952, Alfred Hershey and Martha Chase conducted a series of experiments to prove that DNA was the genetic
material.
 Viruses (T2 bacteriophage) were grown in one of two isotopic mediums in order to radioactively label a specific viral
component.
 Viruses grown in radioactive sulfur (35S) had radiolabelled proteins (sulfur is present in proteins but not DNA).
 Viruses grown in radioactive phosphorus (32P) had radiolabeled DNA (phosphorus is present in DNA but not proteins).
 The viruses were then allowed to infect a bacterium (E. coli) and then the virus and bacteria were separated via
centrifugation.
 The larger bacteria formed a solid pellet while the smaller viruses remained in the supernatant.
 The bacterial pellet was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses
(protein).
 Physical properties of DNA

• According to the Watson and Crick model, the DNA is a double-stranded helix, which consists of two
polynucleotide chains. The two-polynucleotide chain are spirally or helically twisted, which gives it
a twisted ladder-like look.
• Both the polynucleotide strands of DNA have the opposite polarities, which mean that the two strands
will run in the antiparallel direction, i.e., one in 5’-3’ and other in 3’-5’ direction.
• The diameter of ds-stranded DNA helix is 20Å.
• The distance between the two nucleotides or internuclear distance is 3.4Å. The length of DNA helix
is 34Å after a full turn and it possesses 10 base pairs per turn.
• The DNA is twisted in “Right-handed direction”, or we can say in a “Clockwise direction”.
• Turning of DNA causes a formation of wide indentations, i.e., “Major groove”. The distance between
the two strands forms a narrow indentation, i.e., “Minor groove”. The formation of major and minor
grooves result after the DNA coiling and the grooves also act as a site of DNA binding proteins.
 Chemical properties of DNA

• There are four nucleotide bases present in the polynucleotide chain like adenine, guanine, cytosine and thymine.
Adenine and guanine are the two purine bases, which have a single ring structure. Cytosine and thymine are the two
pyrimidine bases, which have the double-ring structure.
• The two strands are joined together by the “Complementary base pairing” of the nitrogenous bases. Therefore, a purine
base will complementarily pair with the pyrimidine base, in which ‘Adenine’ pairs with ‘Thymine’ and ‘Guanine’ pairs
with ‘Cytosine’.
• The nucleotide bases in the polynucleotide strands of DNA will join with each other through a strong hydrogen bond.
• Adenine complementarily pairs with thymine through two hydrogen bonds, whereas guanine complementarily pairs
with cytosine by means of three hydrogen bonds.
• The nucleotide base composition of DNA follows the Chargaff’s rule.
• Polynucleotide strands of DNA consist of three major components, namely nitrogenous bases, deoxyribose sugar and
a phosphate group.
• The backbone of DNA consists of the sugar-phosphate backbone. The sugar-phosphate backbone holds both the
polynucleotide strands of DNA by means of “Phosphodiester bond”. Therefore, the bonding between sugar and
phosphates, i.e., phosphodiester bond and the bonding between nitrogenous bases, i.e., hydrogen bond contributes to
the “DNA Stability”.

Chargaff’s law

 Erwin Chargaff, a biochemist, discovered that the number of nitrogenous bases in the DNA was
present in equal quantities.
 Erwin Chargaff proposed two rules:
 First rule, in any double stranded DNA the number of guanine units equals the number of cytosine
units, and the number of adenine units equals the number of thymine units.
 Second rule, the composition of DNA varies from one species to another.
1) The molar ratio of A to T equals to 1. Similarly, the molar ratio of G to C equals to 1.
2) The sum of the purines (A and G) equals that of the pyrimidines (C and T).
3) The percentage of C+G does not necessarily equal the percentage of A+T
 Nucleoside: Sugar
and nitrogenous
base join to form
nucleoside.
 Nucleotides: are
phosphoric acid
esters of
nucleosides with
phosphate at
position C5’.
 The three
models for
DNA
replication

• Semi-conservative replication. In this model, the two

strands of DNA unwind from each other, and each acts as
a template for synthesis of a new, complementary strand.
This results in two DNA molecules with one original strand
and one new strand.
• Conservative replication. In this model, DNA replication
results in one molecule that consists of both original DNA
strands (identical to the original DNA molecule) and
another molecule that consists of two new strands (with
exactly the same sequences as the original molecule).
• Dispersive replication. In the dispersive model, DNA
replication results in two DNA molecules that are
mixtures, or “hybrids,” of parental and daughter DNA. In
this model, each individual strand is a patchwork of
original and new DNA.
 The Meselson-Stahl experiment

 Meselson and Stahl conducted their famous experiments on DNA replication using E. coli bacteria as a model system.
 They began by growing E. coli in medium, or nutrient broth, containing a "heavy" isotope of nitrogen, 15N15N.
(An isotope is just a version of an element that differs from other versions by the number of neutrons in its nucleus.)
When grown on medium containing heavy 15N15N the bacteria took up the nitrogen and used it to synthesize new
biological molecules, including DNA.
 After many generations growing in the 15N15N the nitrogenous bases of the bacteria's DNA were all labeled with
heavy 15N15N. Then, the bacteria were switched to medium containing a "light" 14N14N isotope and allowed to grow
for several generations. DNA made after the switch would have to be made up of 14N14N, as this would have been the
only nitrogen available for DNA synthesis.
 Meselson and Stahl knew how often E. coli cells divided, so they were able to collect small samples in each generation
and extract and purify the DNA. They then measured the density of the DNA (and, indirectly, its 15N15N and 14N14N)
using density gradient centrifugation.
 This method separates molecules such as DNA into bands by spinning them at high speeds in the presence of another
molecule, such as cesium chloride, that forms a density gradient from the top to the bottom of the spinning tube.
Density gradient centrifugation allows very small differences—like those between 15N15N and 14N14N labeled DNA—
to be detected.

Results of the
experiment
When DNA from the first
four generations of E.
coli was analyzed, it
produced the pattern of
bands shown in the figure
below:
Generation 0
 DNA isolated from cells at the start of the experiment (“generation 0,” just before the switch to 14N14N medium) produced a
single band after centrifugation. This result made sense because the DNA should have contained only heavy 15N15N at that
time.
Generation 1
 DNA isolated after one generation (one round of DNA replication) also produced a single band when centrifuged. However, this
band was higher, intermediate in density between the heavy 15N15N DNA and the light 14N14N DNA.
 The intermediate band told Meselson and Stahl that the DNA molecules made in the first round of replication was a hybrid of
light and heavy DNA. This result fit with the dispersive and semi-conservative models, but not with the conservative model.
 The conservative model would have predicted two distinct bands in this generation (a band for the heavy original molecule and
a band for the light, newly made molecule).
Generation 2
 Information from the second generation let Meselson and Stahl determine which of the remaining models (semi-conservative or
dispersive) was actually correct.
 When second-generation DNA was centrifuged, it produced two bands. One was in the same position as the intermediate band
from the first generation, while the second was higher (appeared to be labeled only with 14N14N).
 This result told Meselson and Stahl that the DNA was being replicated semi-conservatively. The pattern of two distinct bands—
one at the position of a hybrid molecule and one at the position of a light molecule—is just what we'd expect for semi-
conservative replication (as illustrated in the diagram below). In contrast, in dispersive replication, all the molecules should have
bits of old and new DNA, making it impossible to get a "purely light" molecule.
Generations 3 and 4
 In the semi-conservative model, each hybrid DNA molecule from the second generation
would be expected to give rise to a hybrid molecule and a light molecule in the third
generation, while each light DNA molecule would only yield more light molecules.
 Thus, over the third and fourth generations, we'd expect the hybrid band to become
progressively fainter (because it would represent a smaller fraction of the total DNA) and
the light band to become progressively stronger (because it would represent a larger
fraction).
 As we can see in the figure, Meselson and Stahl saw just this pattern in their results,
confirming a semi-conservative replication model.

Taylor’s experiment in Vicia faba (Broad

bean)
 Taylor’s experiment in Vicia faba
(Broad bean)

 Autoradiography experiment in Vicia faba.

 Autoradiography was also utilized by J.H. Taylor and his co-workers for the study of
duplicating chromosomes in the root tip cells of Vicia faba.
 They treated the root tip with radioactive thymidine to label the DNA. Then the root tip was
grown in the normal medium.
 In the first generation of duplication both chromatids were labelled (interpreted as one DNA
double helix in each chromatid, and only one of the two strands labelled). In the second cycle
of duplication, in each chromosome, one of the two chromatids was found to be labelled. This
was interpreted as showing semi-conservative mode of duplication.
 Taylor's experiment is important in so far as it demonstrated the semiconservative mode of
replication in chromosomes of a higher plant.
 DNA replication

 Origin of replication: DNA replication does not start at random locations but at particular sites.
 Replicon: the unit of DNA in which individual acts of replication takes place. It contains an
origin of replication and control units to regulate the process.
Common features of replication origin:
1) Are unique DNA segments that contain multiple short repeated sequences.
2) Origin regions usually contain an AT rich stretch. This property facilitates unwinding of duplex
DNA because less energy is required to melt AT base pair than GC base pair.
 Topoisomerase: is a nuclease that breaks a phosphodiester bond in a DNA strand.
1) Type 1 topoisomerases: produce a transient single strand break.
2) Type 2 topoisomerases: make a transient double strand break in the helix.

Replication comprises

 Primase: which synthesizes a short RNA primers.

 Replication fork: The point at which strands of the parental DNA are separated and
replication is taking place (new nucleotides are being added) is called the
replication fork.
 Helicase: Which opens the duplex at the replication fork to provide a single
stranded template.
 SSB protein (single stranded binding protein): The separated strands are inhibited
from subsequently reannealing by the SSB proteins.
 DNA Ligase: Bonding between fragments and replaced nucleotide.
 DNA Gyrase: is a bacterial enzyme involved in DNA replication and repair. It
belongs to the type II topoisomerase family. It introduces negative supercoils into
DNA to relieve torsional strain during replication.
 Prokaryotic DNA replication

 Replication in prokaryotes is to duplicate their genome into second copy.

Comprises of:
 DNA gyrase
 Single origin of replication
 DNA helicase
 Replication fork (Y shaped structure)
 SSB proteins
 DNA primases (makes 5 to 10 base pair long RNA primer)
 DNA polymerases:
1. DNA polymerase I
2. DNA polymerase II
3. DNA polymerase II
 DNA ligase
 DNA Polymerases in prokaryotes

 DNA polymerase I
Removal of RNA primer and replace it with DNA nucleotide.
 DNA polymerase I,II
Gap filling and DNA repair
 DNA polymerase III
Initiation and elongation of DNA replication
Adds nucleotide from 5’ to 3’ direction.
3’ 5’ direction (lagging strand)
5’ 3’ direction (leading strand)
 Eukaryotic DNA replication

 Genome duplicates prior to cell division.

 There is little change from prokaryotes because
of the size and the structure of the DNA.
 There is a 3-step process:
1) Initiation
2) Elongation
3) Termination

Initiation:
 There is multiple
replication origin.
 DNA helicase- to
unwind DNA
 SSB proteins- To
stabilize the
separated DNA
strand.
 Elongation:

 DNA polymerase α, β , δ, and ε

1) DNA polymerase α (Primase)- Initiates the DNA replication.
Synthesis of RNA primer.
2) DNA polymerase δ, and ε - elongation
 DNA polymerase δ- lagging strand synthesis
 DNA polymerase ε – leading strand synthesis
3) DNA polymerase β- removes the RNA primer. (Similar to DNA
pol1)

Termination:
 Once the leading
strand of a one
replication bubble
meets a lagging
strand of a second
replication bubble,
the replication
process is halted.
 Difference between prokaryotic and
eukaryotic replication:
PROKARYOTE EUKARYOTE
Definition Prokaryotic DNA replication is the process by Eukaryotic DNA replication is the
which a prokaryotic organism duplicates its process by which the eukaryotic
entire genome in order to pass the second genome duplicates prior to cell
copy to a daughter cell. division.
Occurrence Prokaryotic DNA replication is a continuous Eukaryotic DNA replication occurs
process. during the S phase of the cell
cycle.
Location Prokaryotic DNA replication takes place in Eukaryotic DNA replication takes
the cytoplasm. place in the nucleus.
Type of DNA Prokaryotic DNA is circular and double- Eukaryotic DNA is linear and
stranded. double-stranded with ends.
Amount of DNA There is a small amount of Prokaryotic DNA. The amount of eukaryotic DNA is
50 times more than the amount of
prokaryotic DNA.

Difference between prokaryotic and

eukaryotic replication:
PROKARYOTE EUKARYOTE

Origin of Replication Prokaryotic DNA consists of a Eukaryotic DNA consists of multiple

single origin of replication. origins of replication (over 1000).

DNA Polymerases Prokaryotic DNA replication is Eukaryotic DNA replication is

carried out by DNA polymerase I carried by DNA polymerase α, δ,
and III. and ε.

DNA Gyrase DNA gyrase is involved in the DNA gyrase is not required for the
prokaryotic DNA replication. eukaryotic DNA replication
DNA gyrase is not required for the
eukaryotic DNA replication

End Synthesis Prokaryotic DNA does not contain Telomerase is involved in the end
ends. synthesis in Eukaryotic DNA during
the replication.
 Semi discontinuous replication:

 DNA replication is ‘semi-discontinuous’ because one of the strands is

synthesized continuously, while the other strand, discontinuously by the
formation of Okazaki fragments.
 This was discovered in the year 1968 by a Japanese scientist couple -
Reiji Okazaki and his wife Tsuneko Okazaki. The term ‘Okazaki fragments’
has been given after them.
 The leading strand is synthesized continuously in the direction of
replication fork movement. The lagging strand is synthesized in small
pieces (Okazaki fragments) backward from the overall direction of
replication. The Okazaki fragments are then joined by the action of DNA
ligase.

RNA

 RNA or ribonucleic acid is a polymer of nucleotides that is made up of a ribose sugar, a

phosphate, and bases such as adenine, guanine, cytosine, and uracil.
 It plays a crucial role in gene expression by acting as the intermediate between the genetic
information encoded by DNA and proteins.
 RNA has a structure very similar to that of DNA. The key difference in RNA structure is that
the ribose sugar in RNA possesses a hydroxyl (-OH) group that is absent in DNA.
 In both prokaryotes and eukaryotes, there are three main types of RNA – messenger RNA
(mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
 mRNA

 Messenger RNA (abbreviated mRNA) is a type of single-stranded RNA involved in protein synthesis.
 mRNA is made from a DNA template during the process of transcription.
 The role of mRNA is to carry protein information from the DNA in a cell’s nucleus to the cell’s cytoplasm
(watery interior), where the protein-making machinery reads the mRNA sequence and translates each
three-base codon into its corresponding amino acid in a growing protein chain.
 So, we have DNA in our nuclei. And then we have ribosomes and other cellular organelles which translate
DNA. But between the DNA code itself, and the machinery that uses DNA to make proteins, there has to
be a translator. And mRNA is actually the translated form of DNA that the machinery can recognize and
use to assemble amino acids into proteins.

mRNA

 Messenger RNA carries the genetic information copied from DNA in

the form of a series of three-base code words, each of which specifies a
particular amino acid.
 Each codon codes for a specific amino acid, except the stop codons,
which terminate protein synthesis.
 The translation of codons into amino acids requires two other types of
RNA: transfer RNA, which recognizes the codon and provides the
corresponding amino acid, and ribosomal RNA (rRNA), the central
component of the ribosome's protein-manufacturing machinery.
 tRNA

 Transfer RNA (abbreviated tRNA) is a small RNA molecule that plays a key
role in protein synthesis. Transfer RNA serves as a link (or adaptor) between
the messenger RNA (mRNA) molecule and the growing chain of amino acids
that make up a protein. Each time an amino acid is added to the chain, a
specific tRNA pairs with its complementary sequence on the mRNA
molecule, ensuring that the appropriate amino acid is inserted into the protein
being synthesized.
 tRNA

 tRNA is a single RNA chain of 73-93 nucleotides, present in the cytosol and organelles of all living cells.
 tRNA folds into cloverleaf-like secondary structure with well defined stems and loops that make up the
acceptor arm, D arm and loop, anticodon arm and loop, and the T-arm and loop.
 The acceptor stem always has 7 base pairs and 4 single –stranded nucleotides, including an
absolutely conserved CCA sequence.
 The D stem and loop are of variable length, whereas the anticodon stem has 5 nucleotides and the
anticodon loop has 7 nucleotides.
 The variable region usually has 4-5 nucleotides but can contain up to 24 nucleotides.
 Finally, the T stem always has 5 base pairs and the T loop has 7 nucleotides.
 All tRNA share the same secondary structure.
 tRNA are thought to fold into the L-shaped 3D structures necessary to fulfill their function.
 In each cell or organelle, there are 20 families of tRNA, one per each amino acid.
  video

rRNA

 The protein synthesis factories in the cell.

 rRNA molecules are the most abundant form of RNA in the cell.
 80% of RNA molecules
 Different rRNAs present in the ribosomes include small rRNAs and large rRNAs,
which belong to the small and large subunits of the ribosome, respectively.
 rRNAs combine with proteins and enzymes in the cytoplasm to form ribosomes,
which act as the site of protein synthesis. These complex structures travel along the
mRNA molecule during translation and facilitate the assembly of amino acids to
form a polypeptide chain. They interact with tRNAs and other molecules that are
crucial to protein synthesis.
 Function
 Large subunit have
a catalytic role.
(Catalyzes peptide
bond formation)
 18S and 16S rRNA
has recognition
role (correct
positioning of the
mRNA and the
peptidyl tRNA)
 Nucleic acid estimation: Colorimetry

 Colorimetry is the field in which a coloured compound’s concentration in a solution is

measured.
 Calculates the absorption of a certain wavelength of light as a method to calculate a
solution concentration.
 The colorimeter is usually used to measure the concentration of a known solute in a
given solution with the help of the Beer-Lambert law.
 Beer’s law: When monochromatic light passes through a colored solution, the amount
of light absorbed is directly proportional to the concentration (C) of solute in the
solution.
 Lambert’s law: When monochromatic light passes through a colored solution, the
amount of light absorbed is directly proportional to the length (L) or thickness of the
solution.
Nucleic acid estimation: Spectrophotometry

 The spectrophotometer is an instrument which measures the amount of light that

a sample absorbs.
 Traits Colorimeter Spectrophotometer

Monochromator Filter Prism

Wavelength offered Fixed, in visible range of Wide range in UV, visible and
spectrum infrared range of spectrum.

Application It determines the concentration It identifies and quantifies

of compound according to its organic and inorganic
absorbance level. biochemical molecules.
Function Measuring light absorbance Measuring light transmittance
level. level.

Accuracy, sensitivity and Less More

complexity

Sample Large volume needed Small volume needed

Cost Cheaper More costly

DNA ESTIMATION BY
DPA(Diphenylamine) METHOD

 The deoxyribose in DNA in the presence of acid forms β-

hydroxylevulinaldehyde which reacts with diphenylamine to give a blue
colour with a sharp absorption maximum at 595nm.
 The sugar that is linked to purine residues takes part in the reaction
therefore the readout is only from 50% of the total nucleotides.
 This holds true for both known standard and the given unknown sample
therefore the concentration of the unknown sample is directly calculated
from the standard graph.
 Estimation of DNA (DPA method)

 Reagents: DNA standard- 2mg/ml

 DPA reagent- Add 5g DPA in 7 ml of conc. H2SO4. Final volume is made to 500ml by adding
acetic acid.
S.NO Volume of DNA Volume of DNA Water (ml) DPA (ml) Absorption
(mg) (ml) (595nm)
Blank - - 3 6 -

1 1 0.5 2.5 6

2 2 1.0 2.0 6

3 3 1.5 1.5 6

4 4 2.0 1.0 6

5 5 2.5 0.5 6

RNA Estimation orcinol method

 Pentose sugar present in ribonucleic acid reacts with hot acid to form furfural, which on
reaction with orcinol and ferric chloride produces green color. Purine nucleotides are more
reactive than pyrimidine. The green color can be quantitated using UV spectrophotometer
or colorimeter at 665 nm.
Requirements:
1. Standard RNA solution- 200µg/ml in 1 N perchloric acid/buffered saline.
2. Orcinol Reagent- Dissolve 0.1g of ferric chloride in 100 ml of concentrated HCl and add 3.5
ml of 6% w/v orcinol in alcohol.
 CENTRAL DOGMA OF
LIFE AND PROTEIN
SYNTHESIS

Concept of central dogma:

• Central dogma illustrates the flow of genetic information from DNA to RNA
to protein.
• Definition: “Central dogma is the process in which the genetic information
flows from DNA to RNA, to make a functional product protein.“
Terms
• Transcription is the process of copying a segment of DNA into
RNA.
• Translation is the process in which ribosomes in the cytoplasm or
endoplasmic reticulum synthesize proteins after the process of
transcription of DNA to RNA in the cell's nucleus.
• Reverse transcription: The process in cells by which an enzyme
makes a copy of DNA from RNA. The enzyme that makes the
DNA copy is called reverse transcriptase and is found in
retroviruses, such as the human immunodeficiency virus (HIV),
(HTLV-1) Human T-lymphotropic virus-1

Terms:
• Transcription unit: Each transcribed segment of DNA is called
a transcription unit.
• Transcription starts from the first base pair that is called the start point. From this point,
RNA polymerase moves along the template, synthesizing RNA, until it reaches a
terminator sequence.
• The sequences prior to the start point are described as upstream of it; those after the
start point (within the transcribed sequence) are downstream of it.
• Promoter: This is present upstream of the structural gene. It is named by the 5′ end (of
the coding strand which is the 3′ end of the template strand). It has multiple sections to
bind to different transcription factors.
• Structural gene: This is a part of the DNA strand that has a polarity of 3′->5′. This DNA
strand is referred to as the template strand or master strand or antisense, or minus (-)
strand. During transcription, the other strand with a polarity of 5′ is displaced. The non-
template strand which does not participate in transcription is sometimes referred to as
sense or coding strand or plus (+) strand because the genetic code in this strand is similar
to genetic code (based on mRNA), except that uracil is replaced with thymine.
• Terminator: This region is located at the 3′ end (of the coding strand which is the 5′ end of
the template strand) downstream of the structural gene.

Terms:
• Cistrons are segments of genetic material (RNA or DNA) that code for a
functional protein. Monocistronic means one cistron in a transcription
unit, i.e. it contains only one structural gene, coding for a single
polypeptide chain (protein). Each gene contains a separate promoter
region, e.g. in most eukaryotic cells.
• Polycistronic RNA: A messenger RNA that encodes two or more
proteins.
• hnRNA (heterogeneous nuclear RNA): The hnRNA is the collective
term for the unprocessed mRNA (pre-mRNA) molecules in the nucleus. It
is largely comprised of the pre-mRNA molecules that require extensive
processing to become mature mRNA molecules.
 Transcription:
• There are 3 steps:

1) Initiation
2) Elongation
3) Termination
• Main enzyme involved in transcription is RNA polymerase.

RNA polymerase:
1) It copies the information of DNA to a new RNA molecules.
2) RNA polymerase build the strand from 5’ to 3’ adding new nucleotide to 3’ end.
 Initiation:
• RNA polymerase binds to the promoter region on the DNA.
• Promoter region is a guide which tells RNA polymerase where
to attach.
• Once the RNA polymerase attach to the DNA it separates the
DNA strand.
• Only one of the DNA strand is used in transcription which is
known as template strand/ antisense strand/ negative strand.
This strand always runs from 3’ to 5’ direction. This strand is
chosen by the RNA polymerase because synthesis of mRNA
strand is done from 5’ to 3’ direction.

Elongation:
• Here RNA copy is made using DNA template strand.
 Termination
• Terminator sequence in the gene which tells where to stop.
• New pre-mRNA is released from the transcription
complex.
• Pre-mRNA formed is not yet ready to leave the nucleus.
• Post transcriptional changes take place before mRNA is
released into cytoplasm.
 Transcription in prokaryotes:
• RNA polymerase synthesizes all the RNAs in prokaryotes.
• It possesses a core complex with five subunits (ββ′α2ω).
• Two α-subunits assemble the RNA polymerase on the DNA.
• β-subunit adheres with the ribonucleoside triphosphate of nascent mRNA.
• β′-subunit attaches to the DNA template strand.
• The function of the ω-subunit (omega subunit) is not known.
• Sigma protein is the sixth subunit that assembles during the gene transcription and dissembles
during the transcription termination.
• It associates with the core complex of the RNA polymerase to make it biochemically active.
• It aids in promoter recognition, correct binding of the RNA polymerase to the promoter
sequences of the DNA and promotes DNA-unwinding at the start site.
• A sigma protein subunit dissociates after transcription initiation.
 Initiation
• The nucleotide pair in the DNA double helix that corresponds to the site
from which the first 5′ mRNA nucleotide is transcribed is called the +1 site,
or the initiation site. Nucleotides preceding the initiation site are given
negative numbers and are designated upstream. Conversely, nucleotides
following the initiation site are denoted with “+” numbering and are called
downstream nucleotides.
• A promoter is a DNA sequence onto which the transcription machinery
binds and initiates transcription.
1) Prinbnow box (TATA box): This consist of 6 nucleotide bases (TATAAT),
located 10 bases away (upstream) from the starting point of
transcription.
2) The –35 sequence: Base sequence TTGACA which is located about 35
bases upstream from the starting point of transcription.
 Elongation
• As the holoenzyme, RNA polymerase recognizes the
promoter region, the sigma factor is released and
transcription proceeds.
• RNA is synthesized from 5' end to 3' end antiparallel to
the DNA template.
• The double helical structure of DNA unwinds as
the transcription goes on, resulting in supercoils.
• The problem of supercoil is overcome by
topoisomerases.
 Termination:
Rho independent termination:
• Termination in this case is brought about by the formation of
hairpins of newly synthesized RNA.
• This is because of complementary sequence base pair.
• RNA polymerase slows down and pauses due to the formation
of a hairpin loop structure.
• Hair pin loop structure is followed by continuous string of U
nucleotides which destabilises the binding of RNA polymerase
on m-RNA template.
• RNA polymerase dissociate and transcription is terminated.
Termination
• Rho dependent termination:
• A specific protein named rho factor binds to the
growing RNA (and not to RNA polymerase) or weakly
to DNA and in the bound state it acts as ATPase
(molecular motor) and terminates transcription and
release RNA.
• The rho factor is also responsible for the dissociation
of RNA polymerase from DNA.
Eukaryotic RNA polymerase
•RNA polymerase I – Ribosomal RNA
•RNA polymerase II – Messenger RNA
•RNA polymerase III – Transfer RNA
Promoter site:
1) Hogness box/ TATA box – about 25 nucleotides
upstream
2) CAAT box- 70 to 80 nucleotides upstream
• RNA polymerase II along with many other proteins
called as transcription factors are required for the
initiation of transcription.
TF II D binds to the TATA element on the
promoter.
 TF II D has a protein called TBP (TATA binding
protein) which binds TATA sequence.

Once TBP binds TATA sequence it bends DNA by 80

degree. This bending facilitates binding of other
transcription factors.
TF II A helps in stabilizing the binding of TF II D with the promoter.
TF II B interacts with TBP and promoter region downstream to TATA region.
TF II B helps in the recruitment of RNA pol II on the promoter.

RNA polymerase cannot bind to the promoter

region on its own. TF II F helps the RNA
polymerase to bind to the promoter.
Next TF II E binds to the pre initiator
complex. This helps in binding of TF II H.

TH II H is a large molecule containing 9

subunits.
ATPase activity: acts like helicase which converts preinitiation
complex to open complex.
Kinase activity: Phosphorylates C terminal domain/ tail of RNA
polymerase II leading to promoter escape and elongation.
Transcription factor which help in elongation is called
elongation factor.
TFEb- helps in phosphorylation and thus helps in
elongation.
TF II S- helps to increase the speed of transcription
where it is slow and it does not allow RNA
polymerase to pause and move on.

5' capping:
RNA triphosphatase: remove the terminal
gamma phosphate of nucleotide.

Guanylyl transferase: carries out reaction

between beta phosphate of nucleotide and
alpha phosphate of GTP.
Once guanine is attached, methyl transferase
attach a methyl group to guanine nucleotide.

5' capping helps in recruitment of mRNA on

the ribosome for the initiation of translation.
Termination : When the RNA polymerase reach the
end of the gene, the C terminal domain of RNA pol
II reacts with 2 protein- CstF and CPSF

CstF cleaves the mRNA and after that it

dissociates.
CPSF recruits poly A polymerase which adds about
200 adenine residues at 3' end giving rise to poly A
tail. Poly A polymerase uses ATP for this process.

Once poly A tail is formed poly A binding

protein binds to poly A tail. This protein
prevents the degradation of poly A tail.
Poly A tail/ Polyadenylation
• Poly A tail allows the mature messenger RNA
molecule to be exported from the nucleus and
translated into a protein by ribosomes.

Once poly A binding protein binds to the

tail end of mRNA, CPSF is released.
 Split gene:
• The coding regions containing actual information of the genes (exons) of most
eukaryotic genes are interrupted by few to several non-coding sequences called
introns which are spliced out after transcription such genes are called split
genes.
• Introns are the interrupting sequences that do not code for any protein
products.
• Each interrupted gene or the split gene will have an exon at the beginning as well
as the end.

RNA splicing
RNA splicing is a process in molecular biology where a newly-
made precursor messenger RNA (pre-mRNA) transcript is
transformed into a mature messenger RNA (mRNA).

It works by removing all the introns (non-coding regions

of RNA) and splicing back together exons (coding
regions).

For many eukaryotic introns, splicing occurs in a series of

reactions which are catalyzed by the spliceosome, a
complex of small nuclear ribonucleoproteins (snRNPs).
 Concept of reverse transcription
• Some of the viruses- known as retroviruses possess RNA as the genetic material. The enzyme RNA
dependent DNA polymerase- or simply reverse transcriptase- is responsible for the formation of DNA
from RNA. This DNA is complementary (cDNA) to viral RNA and can be transmitted into host DNA.
• Reverse transcriptase is central to the infectious nature of retroviruses, several of which cause disease in
humans, including human immunodeficiency virus (HIV), which causes acquired immunodeficiency
syndrome (AIDS), and human T-cell lymphotrophic virus I (HTLV-I), which causes leukemia.
• Reverse transcriptase is also a fundamental component of a laboratory technology known as reverse
transcription-polymerase chain reaction (RT-PCR), a powerful tool used in research and in
the diagnosis of diseases such as cancer.
• Retroviruses consist of an RNA genome contained within a protein shell that is enclosed in
a lipid envelope.
• The retrovirus genome is typically made up of three genes: the group-specific antigen gene (gag), the
polymerase gene (pol), and the envelope gene (env).
• The pol gene encodes the three enzymes—protease, reverse transcriptase, and integrase—that
catalyze the steps of retroviral infection.
• Once a retrovirus is inside a host cell (a process mediated by protease), it takes over the host’s genetic
transcription machinery to construct a DNA provirus. This process, the conversion of retroviral RNA to
proviral DNA, is catalyzed by reverse transcriptase and is necessary for proviral DNA insertion into host
DNA—a step initiated by the integrase enzyme.

Function:
Three genes:
• Group-specific antigen gene (gag): Codes for the matrix, capsid (proteinaceous capsule)
and structural core proteins of the virus.
• Polymerase gene (pol): encodes the three enzymes—protease, reverse transcriptase, and
integrase
• Envelope gene (env):Encodes the protein forming viral envelope
 Function

Reverse
Protease: Hydrolyse Integrase: Catalyzes
transcriptase:
the peptide the insertion of viral
Responsible for the
bonds present in DNA into host
formation of
proteins chromosomal DNA.
DNA from RNA
Genetic code
• genetic code, the sequence of nucleotides in deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) that determines the amino acid sequence of proteins.
• There are 64 possible codons, three of which do not code for amino acids but
indicate the end of a protein. The remaining 61 codons specify the 20 amino acids
that make up proteins.
• The AUG codon, in addition to coding for methionine, is found at the beginning of
every mRNA and indicates the start of a protein.
 • Triplet code
• Unambiguous and Universal
• Degenerate code
Properties of • Nonoverlapping code
genetic code
• Punctuationless
• Start and Stop Codons
• Polarity
Triplet code
• Genetic code is made up of three consecutive nucleotides called codons on the
mRNA and is read from 5' to 3' direction.

Punctuationless
• There are no punctuation marks like comma, colon or semi-colon.
 Nonoverlapping code
• The code is read sequentially in a group of three and a nucleotide which becomes
a part of triplet never becomes part of the next triplet.
• For example
• 5’-UCU-3’ codes for Serine
• 5’-AUG-3’ codes for methionine

Polarity
• Each triplet is read from 5’ → 3’ direction and the beginning base is 5’ followed by the base in
the middle then the last base which is 3’. This implies that the codons have a fixed polarity and
if the codon is read in the reverse direction, the base sequence of the codon would reverse and
would specify two different proteins.
 Degenerate code

Start and Stop Codons

• Generally, AUG codon is the initiating or start codon. The polypeptide chain starts either with
eukaryotes (methionine) or prokaryotes (N- formylmethionine).
• On the other hand, UAG, UAA and UGA are called as termination codons or stop codons. These
are not read by any tRNA molecules and they never code for any amino acids.
 Unambiguous and Universal

CODON ANTICODON
A codon is the triplet sequence in An anticodon is the corresponding
the mRNA transcript which triplet sequence on the transfer
specifies a corresponding amino RNA which brings in the specific
acid. amino acid to the ribosome during
translation.
PROKARYOTE

Translation in prokaryotes
•Initiation
•Elongation
•termination
Initiation is carried out by 3 initiation factors
IF 1, IF 2, IF 3

IF3 binds to the 30s subunit of the

ribosome at E site
IF3 separates the 50s and 30s subunits

IF1 with GTP binds to the A site of the 30s

subunit. IF3 blocks the E site and IF1 blocks
the A site.
IF2 along with charged N formyl methionine
tRNA binds to P site.

This structure formed is called 30s

initiation complex.
Binding of IF2 to P site causes IF3 to
release from E site.

Once IF3 is released 50s subunit along

with mRNA binds to the 30s subunit.
GTP associated with IF1 is hydrolysed to
GDP by IF2

GTP associated with IF1 is hydrolysed to

GDP by IF2
 Once hydrolysed the IF1 is released from A
site.

Finally, IF2 is also released from ribosome.

This complex formed is called 70s initiation
complex.

Elongation factors are :

GTP bound EF-Tu brings the charged amino acid
(amino acyl tRNA) to the A site of the ribosome.

Once EF-Tu binds the GTP is hydrolysed

to GDP.
Finally, EF-Tu GDP is released from the
ribosome. EF-Ts helps in recycling of GDP by
GTP.

Next a peptide bond formation between

two amino acid takes place.
GTP bound EF-G comes to A site where
the GTP gets hydrolysed to GDP.

tRNA carrying amino acid gets shifted

from A to P site.
Once it gets shifted EF-G is released
Once the stop codon reaches the A site the
translation machinery proceeds for
termination.

There are three release factors in

termination
RF1 occupies A site which has 3 amino acid
and this helps in release of polypeptide chain
from the ribosome.

This is triggered when GDP bound RF3

binds to RF1.
Peptide chain and the RF1 is released.

GDP is replaced by GTP which releases

RF3
RRF (ribosome recycling factor) binds at A
site.

EF-G with GTP binds to A site which causes

RRF to release the uncharged tRNA from E
and P site.
 EF-G GTP gets hydrolysed to GDP

IF3 binds to 30s subunit at E site which will causes

the dissociation of translation machinery.
Eukaryotes

Three initiation factors eIF1, eIF1a, eIF3

binds to the 40s subunit.
Then comes the charged tRNA with
methionine which with the help of eIF2 with
bound GTP helps the tRNA to be carried to the
P site on 40s subunit.

Then eIF5B with GTP binds to the 40s complex

forming 43s preinitiation complex.
Then you have mRNA strand with 5’ cap and
poly A tail and with few secondary structures.
It will have an AUG codon from where the
translation will start.

Then comes another three factors eIF4A,

eIF4E, eIF4G.
eIF4E binds to 5’ end of mRNA. Then
eIF4G binds to eIF4E.

To eIF4G poly A binding protein (PABP)

binds.
eIF4A acts like helicase and remove the
secondary structure in tRNA.

PABP interacts with poly A tail.

Now 43s preinitiation complex interacts with
mRNA at the 5’cap of mRNA and forms 48s
complex.

Then ribosome will scan for AUG. Once the

AUG is reached the GTP hydrolysis occur and
all the factors leave the 40s subunit.
60s subunit is recruited to 40s subunit
forming 80s subunit.

eEF1 alpha with bound GTP brings the

charged tRNA to A site. Translocation is done
by eEF2 factor
eRF1 recognises stop codon on mRNA

Once eRF1 binds the tRNA will not be able to

bind the mRNA and thereby terminating the
process and releasing the polypeptide chain.
Group of genes that regulate the
metabolism of lactose.

• E.coli a bacteria which resides in our gut and it

eats whatever you eat. Glucose that is released
is used as energy by these E.coli in your gut.
• Lactose is a disaccharides which has two simple
sugar- Glucose and galactose.
• Bacteria will not break down lactose without
reason because:
1) It needs energy to break down disaccharides.
2) Glucose is readily available so no need to break.
• Lactose becomes essential in the absence of
glucose.
• So there is a switch kind of action in bacteria
that can regulate this process.
 Lac operon gene in E.coli

There are two main regions: structural genes and

regulatory gene. Structural genes code for enzymes
that break down lactose. Regulatory genes regulates
or controls structural genes.
Trp operon
 Difference between lac and trp operon
Lac operon Trp operon

Operon type Inducible Repressible

No. of genes 3 5

Function of operon Breakdown lactose Synthesize

gene tryptophan
Repressor gene Lac I Trp R