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Biology by Dr. Vani Sood

Molecular basis
Genetics
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NATURE OF GENE, ITS EXPRESSION
AND REGULATION
Requirements for a genetic material

❖ Replicates itself accurately during cell cycle and duplication

❖ Stable structure so that heritable changes or mutations occur

rarely

❖ Carries all the necessary biological information

❖ Forms the link between two generations in transferring the

hereditary information
DNA: the genetic material

▪ Deoxyribonucleic acid
▪ 1868: Friedrich Miescher described DNA
▪ 1928: Frederick Griffith called it “transforming principle” while
working on the bacterial strain Streptococcus pneumoniae
▪ Sutton and Boveri: genetic information is passed from generation
to generation by chromosomes
Griffith’s experiment
▪ 1944: Avery, MacLeod and McCarty showed that the transforming principle
consists entirely of DNA.
▪ Heat killed DNA of S strain extracted and injected into R strain, in vitro and
allowed to grow in culture medium.
▪ Colonies: S type proving that the transforming principle is DNA that converted
avirulent R into virulent S type.
▪ Extract: treated with DNAase (an enzyme that kills DNA) - transforming ability
was lost.
▪ Proteases (enzyme that destroys proteins): no effect
▪ Hence proved: DNA and not the proteins is the genetic material
 RNA: the genetic material

▪ Ribonucleic acid
▪ Tobacco mosaic virus uses RNA instead of DNA as its sole
genetic material.
▪ RNA: surrounded by a protein coat which protects it from
the action of ribonuclease and also helps it in penetrating
plant cells thereby causing infection.
Structure of DNA
Watson and Crick, 1953
DNA is made up of repeating molecules called
NUCLEOTIDES
Phosphate
Group

O 5
O=P-O CH2
O
O
N
Nitrogenous base
1
C4 C (A, G, C, or T)
Sugar
(deoxyribose)
C3 C2
Watson & Crick proposed…
•DNA had specific pairing between the nitrogen bases:
ADENINE – THYMINE
CYTOSINE – GUANINE

• PURINES • PYRIMIDINES
1. Adenine (A) 3. Thymine (T)
2. Guanine (G) 4. Cytosine (C)
Arrangement of nucleotides

5´ 3´
“Rungs of ladder”

Nitrogenous
Base (A,T,G or C)

“Legs of ladder”

Phosphate &
Sugar Backbone

3´ 5´
Advantages of Double-Stranded Nature of DNA

Forms a stable structure

Hydrophobic bases stack on top of one another away from
solvent
Charged phosphate backbone is on the outside accessible
to solvent
Each strand can serve as the template
For a new strand of DNA (replication)
For an RNA molecule (transcription)
 DNA Double Helix
5 O 3

3 O
P 5 P
5 O
1 G C 3
2
4 4
2 1
3 5
O
P P
5
T A 3
O

O
5
P 3 P
Double stranded DNA
forms a double helix
 Chargaff’s Rule
• Adenine must pair with Thymine
• Guanine must pair with Cytosine

A+G = T+C
A=T; G=C

T A
Hydrogen bonds

G C
Forms of DNA-
Under different conditions of isolation, purification, and crystallization ,
several forms of DNA have been recognized they are A , B , C , D and Z
DNA.
At the time watson and crick performed their analysis two forms namely A
– DNA and B – DNA were known.
Watson and crick analysis were based on X-Rays studies on the B- DNA.
Z- DNA was discovered by andrew wang and alexander rich in 1979.
A , B , C, and D – DNA are right handed helices where as Z- DNA is left
handed helix with 12 base pairs pairs per turn.
 A- DNA

Right-handed
Distance between two
complete turns: ~28.2Å
Distance between two
neighboring base pairs: 2.56Å
Base pairs per turn: 11
Diameter: 23Å
 DNA Forms - B-DNA Helix
Two strands wind about each other in a right-handed
manner
Diameter: ~20Å
Bases per turn: 10
Distance between two complete turns: ~34Å
Distance between two neighboring base pairs: 3.4Å
The intertwined strands make two grooves of different
widths, referred to as the major groove and the minor
groove
 Z- DNA

Named because its bases seem to zigzag.  

Z DNA is left-handed. 
One turn spans: 45Å
Number of base pairs per turn: 12 
Diameter: 18Å
Gene expression

Process by which information from a gene is used in the synthesis

of a functional gene product
Mechanism by which a gene is able to express itself in the
phenotype of an organism
Steps include: transcription, RNA splicing, translation, and
post-translational modification of a protein
DNA: master molecule which directs gene expression
Gene expression
DNA replication: making exact copies
Types of DNA replication:
Replication of DNA:

One property of the genetic material necessary for its function is the ability to
replicate (reproduce) itself.
After it was established that DNA is the genetic material, attention turned toward
how DNA was replicating in living organisms.
The nature of base pairing meant that if the two strands of a DNA molecule were
separated, they could each serve as a template for the creation of a complementary
strand by bringing in individual nucleotides to base pair with their complementary
base on the template, and joining the new nucleotides together.
Thus, each DNA molecule after replication would consist of one of the original
strands plus one newly synthesized strand.
This model of DNA replication is called semiconservative.
Semiconservative was not the only model of DNA replication, however.
Other proposed models included conservative replication and dispersive
replication.
Conservative replication proposed that after replication, one DNA molecule
consists entirely of newly synthesized DNA whereas the other molecule is entirely
original DNA.
Types of replication-
Dispersive replication suggested that each DNA molecule after replication might
consist of segments of new and old DNA interspersed.
Experiment and Results:

E. coli grown on 15 n isotope of nitrogen.

These bacteria got completely labeled.
The labeled bacteria were then shifted to fresh medium having 14 n nitrogen.
Now DNA was checked through density gradient centrifugation using caesium
chloride.
DNA of the first generation was hybrid or intermediate 15 N and 14N and other 14
n.
The second generation contains two types of DNA , 50% light ( n 14 n 14) and 50%
intermediate ( 15 n 14 n)
The third generation of bacteria after 60 mins contained two types of DNA , 25 %
intermediate ( n15 n 14) and 75% light ( n14 n 14 ) in 1:3 ratio.
Difference-
Conservative replication would produce one DNA molecule containing heavy
nitrogen and one molecule containing light nitrogen, so there would be two
different densities.
Dispersive replication would produce a single intermediate density, just like
semiconservative but over time it is seperated as there would be no pure varieties.
1958: Matthew Meselson & Frank Stahl’s Experiment
Requirements for DNA replication

1. A DNA template

2. A primer: dNTPs: dATP, dTTP, dGTP, dCTP

(deoxyribonucleotide triphosphates)

3. DNA polymerase

4. Mg 2+ (optimizes DNA polymerase activity)

DNA template-
DNA replication. The double helix is unwound and each strand acts as
a template for the next strand.
Bases are matched to synthesize the new partner strands.
Primer:

primer is a strand of nucleic acid is a strand of nucleic acid that serves as a

starting point for DNA synthesis.
In most cases of DNA replication, the primer for DNA synthesis and
replication is a short strand of RNA.
Activation of deoxyribonucleotides:

They occur freely inside the nucleoplasm, there are four types-
1. deoxyadenosine triphosphate.
2.deoxyguanosine.
3.deoxycytidine.
4. deoxythymidine.
They are first phosphorylated and changed into active forms with three phosphate
residues instant of one and then they act as substrate and provide energy for
polymerization of nucleotides.
DNA polymerases-
DNA polymerasesDNA polymerases are a family of enzymes that carry out all
forms of DNA replication.
However, a DNA polymerase can only extend an existing DNA strand paired
with a template strand; it cannot begin the synthesis of a new strand.
To begin synthesis, a short fragment of DNA or RNATo begin synthesis, a short
fragment of DNA or RNA, called a primer, must be created and paired with the
template DNA strand.
MOA-
DNA synthesis always occurs in the 5’ 3’ direction.
The mechanism of DNA replication is very similar in most organisms.
Difference exist only with respect to the enzymes and protein involved.
In prokaryotes such as E. coli , two enzymes DNA polymerase 1 and 3 are
responsible for DNA synthesis.
In eukaryotes DNA is replicated by five DNA polymerases ( alpha , beta , gamma
, delta , epsilon) type 2.
▪ DNA polymerase III Roger Kornberg
◆ main DNA builder
▪ DNA polymerase I (Kornberg enzyme)
◆ editing, repair & primer removal

Arthur Kornberg
Type of DNA polymerases (Eukaryotes)

1. α (alpha): nuclear, DNA replication, no proof reading

2. β (beta): nuclear, DNA repair, no proof reading
3. γ (gamma): mitochondria, chloroplast, DNA repl., proof reading
4. δ (delta): nuclear, DNA replication, proof reading; isolated from
calf thymus and rabbit bone marrow
5. ε (epsilon): nuclear, proof reading
Dna replication in S phase.

Proof reading????
As the cell grows and divides, it progresses through stages in the cell cycle; DNA
replication occurs during the S phase (synthesis phase).
The progress of the eukaryotic cell through the cycle is controlled by cell cycle
checkpoints.
The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic cells
enter the process of DNA replication and subsequent division.
Cells that do not proceed through this checkpoint are remain in the G0 stage and
do not replicate their DNA.
 Major steps/enzymes for DNA replication

✔ Segments of single-stranded DNA are called template strands

✔ Initiator proteins and DNA helicase binds to the DNA at the
replication fork and untwist the DNA using energy derived from ATP
Unwinding-
Helicase acts at the ori site.
Helicase unwinds by action of breaking the weak hydrogen bonds between paired
bases.
The separated strands are stabilized by means of single stranded binding proteins.
It prevents the double helix from reforming.
Unwinding causes tension in the uncoiled part by forming super coils.
Gyrase- introduce negative supercoils.
✔ Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.
✔ It releases the tension caused by unwinding.
Difference in unwinding-
Bacterial and viral DNA has a single origin of replication but in eukaryotic DNA
there are a number of origins of replication.ie multireplionic.
In prokaryotic DNA there is a single replicating unit or replicon and various
replicons in eukaryotes.
In eukaryotes the whole dna does not open in one stretch due to very high energy
requirement but the point of separation proceeds slowly towards both the
directions.
It gives a Y shaped structure called replication fork.
Priming by rna polymerase/primase-
✔ Rna primer- its necessary for initiation of new dna chains.
Rna primer-

It’s a small strand of RNA which is synthesized at the 5’ end of new dna strand with
the help of DNA specific RNA polymerase enzyme called primase.
I.e. primase with the help of helicase synthesizes RNA primer.
It is specific.
Rna primer is formed on the free end of one strand and fork end of the other strand.
Formation of rna primer constitutes the initiation phase of dna synthesis because
without the presence of RNA primer , dna polymerase cannot add nucleotides.
Primosome:

✔ Primase next binds to helicase producing a complex called a

primosome.
✔ This exists in x 174 phage bacteria and some other prokaryotes.

✔ Primase synthesizes a short RNA primer of 4-12 nucleotides, to which

DNA polymerase III adds nucleotides in eukaryotes.
✔ Polymerase III adds nucleotides to the 3’ end (replication proceeds
in 5’ to 3’ direction) on both strands beginning at the RNA primer.
✔ The RNA primer is removed and replaced with DNA by
exonuclease polymerase I, and the gap is sealed with DNA ligase.
✔ Single-stranded DNA-binding (SSB) proteins (>200) stabilize the
single-stranded template DNA during the process.
Replication: 1st step
Unwind DNA
Helicase enzyme
unwinds part of DNA helix
stabilized by single-stranded binding proteins (SSB proteins)
helicase

single-stranded binding proteins replication fork

 Replication fork / Replication bubble

3′ 5′

5′ 3′

DNA polymerase III

leading strand
5′
3′ 3′ 5′
5′ 5′
5′ 3′ 3′
lagging strand

5′

3′ leading strand
growing
3′ replication fork 5′
5′ growing
replication fork 5′
leading strand 3′
lagging strand
3′
5′
5′ 5′
Origin of Replication (Ori; Prokaryotes)
✔ Begins with double-helix denaturing into single-strands thus exposing the bases.
✔ Exposes a replication bubble from which replication proceeds in both directions.
Replication: 2nd step

▪ Build daughter DNA strand

◆ add new complementary bases
◆ DNA polymerase III

DNA
Polymerase III
Energy of Replication
The nucleotides arrive as nucleosides
DNA bases with P–P–P
P-P-P = energy for bonding
DNA bases arrive with their own energy source for bonding
bonded by enzyme: DNA polymerase III

ATP GTP TTP CTP

That is the nucleosides are first phosphorylated and changed to active forms
which have three phosphate residues.
Enzyme phosphorylase is required along with energy.
These triphosphates of bases serve dual purpose, they act as substrate as well
as provide energy for polymerization of nucleotides.
Starting DNA synthesis: RNA primers

Limits of DNA polymerase III

◆ can only build onto 3′ end of an existing
DNA strand 5′

3′ 5′ 3′
5′
3′
3′ 5′

growing 3′ primase
5′ replication fork DNA polymerase III

RNA 5′

RNA primer 3′

◆ built by primase
◆ serves as starter sequence for DNA
polymerase III
Leading & Lagging strands

Unwinding of any single DNA replication fork proceeds in one

direction.

The two DNA strands are of opposite polarity, and DNA

polymerases only synthesize DNA 5’ to 3’ strand.
As it adds them on 3’ end.

Solution: DNA is made in opposite directions on each template.

Leading and lagging strand:
leading strand template is the strand of DNA being replicated continuously.
It is the strand that is being continuously polymerized towards the replication
fork.
All DNA synthesis occurs 5'-3'.
The original DNA strand must be read 3'-5' to produce a 5'-3' nascent strand.
The leading strand is formed as a polymerase "reads" the template DNA and
continuously adds nucleotides to the 3' end of the elongating strand.
This polymerase is DNA polymerase IIIThis polymerase is DNA polymerase III
(DNA Pol III) in prokaryotesThis polymerase is DNA polymerase III (DNA Pol III)
in prokaryotes and presumably Pol εThis polymerase is DNA polymerase III
(DNA Pol III) in prokaryotes and presumably Pol ε in eukaryotes.
Lagging strand:
The lagging strand template The lagging strand grows in the direction
opposite to the movement of the growing fork.
It grows away from the replication fork and it is synthesized discontinuously.
Because the strand is growing away from the replication fork, it needs to be
replicated in fragments because the Primase (that adds the RNA primer) has to
wait until the fork opens to be able to put the primer
These fragments of DNA produced on the lagging strand are called Okazaki
fragments.
The orientation of the original DNA on the lagging strand prevents continual
synthesis.
As a result, replication of the lagging strand is more complicated than replication of
the leading strand.
•Leading strand synthesized 5’ to 3’ in the direction of the
replication fork movement.
- continuous
-requires a single RNA primer
•Lagging strand synthesized 5’ to 3’ in the opposite direction.
- semi discontinuous
- requires many RNA primers, DNA is synthesized in short
fragments (Okazaki fragments).reiji okazaki ( 1968)
Lagging strand
◆ Okazaki fragments
◆ joined by ligase
▪ “spot welder” enzyme
5′

a gments
k i f r
Ok aza 3′ 5′ 5′ 3′
5′
3′
5′
Lagging strand
3′
ligase
growing 3′
replication fork
5′
Leading strand
3′
✔ 5′

3′
Leading strand DNA polymerase III
◆ continuous synthesis
Looping for formation of okazaki fragments on lagging
strand

The lagging strand template bends

around 180° to present itself to the
DNA polymerase enzyme in the same
physical direction as the leading strand
 Replacing RNA primers with DNA
DNA polymerase I
◆ removes sections of RNA primer and replaces
with DNA nucleotides
DNA polymerase I
5′

3′

3′
5′ ligase
growing 3′
5′ replication fork

RNA 5′

3′
But DNA polymerase I still can
only build onto 3′ end of an existing
DNA strand
Editing & proofreading DNA

▪ DNA polymerase I
◆ proofreads & corrects
◆ repairs mismatched bases
◆ removes abnormal bases
▪ repairs damage
throughout life
Dna polymerases-
Prokaryotes have three major types of DNA synthesizing enzymes called DNA
polymerases 3 , 2 , 1.
All of them add nucleotides in the 5’ to 3’ direction on 3’ to 5’ strand.
They also possess 3’ to 5’ exonuclease activity, i.e. the RNA primer is removed and
new DNA is added.
DNA pol 3 is mainly involved in dna replication ( addition and polymerisation of new
bases)
Pol 1 is a major repair enzyme.
Pol 2 is a minor repair enzyme.
Pol 1 also has 5’ to 3’ exonuclease activity that is nick translation.
In eularyotes 5 types of DNA polymerases are found.
Alpha, beta, gamma, delta abd epsilon.
The major ones are alpha , delta, epsilon.
Polymerases alpha , delta and epsilon are major.
Polymerase delta is involved in the replication of the leading strand.
Polymerase epsilon is involved in the synthesis of the lagging strand.
Pol alpha forms a complex with primase.
Pol beta is the repairing enzyme.
DNA LIGASE
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond
 Chromosome erosion
All DNA polymerases can
only add to 3′ end of an
existing DNA strand DNA polymerase I
5′

3′

3′
5′
growing 3′
5′ replication fork DNA polymerase III

RNA 5′

Loss of bases at 5′ ends

3′
in every replication
◆ chromosomes get shorter with each replication
Telomeres
Repeating, non-coding sequences at the end of
chromosomes = protective cap

5′

3′

3′
5′
growing 3′ telomerase
5′ replication fork

5′

Telomerase TTAAGGG TTAAGGG TTAAGGG 3′

◆ enzyme extends telomeres

◆ can add DNA bases at 5′ end
 Short overall summary
DNA
polymerase III lagging strand
DNA
polymerase I
3’
Okazaki primase
fragments 5’
5’ ligase
3’ 5’ SSB

3’ helicase

DNA
polymerase III
5’
leading strand
3’
direction of replication
SSB = single-stranded binding proteins
Model of DNA replication
Replication of circular DNA (Prokaryotes)
1. Two replication forks result in a theta-like (θ)
structure.

2. As strands separate, positive supercoils form

elsewhere in the molecule.

3. Topoisomerases relieve tensions in the

supercoils, allowing the DNA to continue to
separate.
 Checkpoints in eukaryotic replication
Copying each eukaryotic chromosome during the S phase of the cell
cycle presents some challenges:
Major checkpoints in the system
1.Cells must be large enough, and the environment favorable.
2.Cell will not enter the mitotic phase unless all the DNA has
replicated.
3.Checkpoints in the system include proteins call cyclins and enzymes
called cyclin-dependent kinases (Cdks).
 Central dogma of molecular biology
▪ One way flow of information from DNA to mRNA and then to protein is
called the Central Dogma
▪ Proposed by F.H.C. Crick in 1958
▪ Two scientists: H. Temin and D. Baltimore
▪ In RNA tumour virus, formation of DNA on RNA template occurs
▪ Called Reverse Transcription or Temenism
▪ Occurs in the influence of the enzyme reverse transcriptase
▪ Retroviruses like HIV
The Flow of Genetic Information
The Genetic Code
• Information is stored in the sequence of nucleotides in the DNA
• The “language” of DNA is made of “words” three bases long called
codons.
• Each codon codes for a specific amino acid in a protein

With four biochemical letters, A,U,G,C a

triplet code based on three biochemical letters
or nucleotide bases could make up to 4x4x4 =
64 codons
Characteristics of the Genetic Code

• non-overlapping
• degenerate; one amino acid coded by more than one codon
• commaless
• triplet
• basically universal over all living species
• polar
• no punctuation
• defines a reading frame
 UAA
UGA
UAG

AUG

61 sense codons
3 nonsense codons
Genes and enzymes-
Archibald garrod (1902) was the first person to hint that
genes operate through enzymes.
Alkaptonuria was caused by a pair of inherited recessive
enzymes.( inborn errors of metabolism)
Alkapton or homogenistic acid is produced in human
beings during metabolism of amino acids phenylalanine
and tyrosine.
Its catabolised by an oxidase enzyme to produce co2 and
h2o.
Alkapton oxidase enzyme absence causes homogenistic
acid to accumulate In the body.
Part of it is excreted in the urine- homogenistic acid.
On standing the acid gets oxidized to form a black or brown product.
Although Garrod published a book and several papers on the subject, his work
was generally ignored until the early 1940's, when it was rediscovered by the
American geneticists, George Beadle and Edward Tatum.
Alkaptonuria-
One gene one enzyme hypothesis-
It’s a hypothesis put forward by beadle and tatum ( 1948).
It states that a gene controls a structural and functional trait through controlling
the synthesis of a specific protein or enzyme formed by the latter.
Beadle and Tatum set out to provide experimental proof of the connection
between genes and enzymes.
They hypothesized that if there really was a one-to-one relationship between genes
and specific enzymes, it should be possible to create genetic mutants that are
unable to carry out specific enzymatic reactions.
To test this theory, they exposed spores of Neurospora crassa (a bread mold)
( phototrpohs)to X-rays or UV radiation and studied the resulting mutations.
(auxotrophs).
The mutant moulds had a variety of special nutritional needs. Unlike their
normal counterparts, they could not live without the addition of particular
vitamins or amino acids to their food.
For example, normal Neurospora requires only one vitamin (biotin), but
mutants were created that also required ornithine, citrulline, arginine.
TRANSCRIPTION
▪ First step in the gene expression and involve copying DNA into RNA.
▪ Transcription: chemically and enzymatically, very similar to DNA
replication
RNA made of ribonucleotides
RNA polymerase catalyzes the reaction; (no primer)
Synthesized RNA does not remain base-paired to the template DNA
strand
Transcription selectively copies only certain parts of the genome and
makes one to several hundred, or even thousand, copies of any given
section of the genome. (replication must copy the entire genome and do
so once every cell division)
Antisense or template strand of DNA-
The process of copying information from the antisense or template strand of the
DNA into RNA is called transcription.
It is meant for taking the coded information from DNA to the site where it is
required for protein synthesis.
Principle of complimentarity is used along with an exception.
Only the TEMPLATE strand of the dna is transcribed, as it will provide us with
two copies of proteins.
One with the correct sequence and the other with reverse sequence..
If two complimentary strands of RNA are produced simultaneously , they would
have a tendency to produce double stranded RNA resulting in non translation.
Transcription unit-

The segment of DNA that takes part in transcription is called transcription unit.
It has three components- a promoter, the structural gene and the terminator.
Besides a promoter eukaryotes also require an enhancer.
PROMOTER- its located upstream on structural gene.
It’s the 3’ end of template strand and 5’ end of coding strand.
TERMINATOR- its present downstream of a strand.
Promoter region:
It has different parts for attachment to various transcription factors.
In many cases the promoter has an AT rich region called TATA box.
The area has a groove to which specific protein components can combine.
Now what is transcription factor????
Transcription factor?????

is a proteinis a protein that binds to specific DNA sequencesis a protein

that binds to specific DNA sequences, thereby controlling the flow (or
transcription) of genetic information from DNA to mRNA.
A defining feature of transcription factors is that they contain one or more
DNA-binding domains (DBDs), which attach to specific sequences of DNA
adjacent to the genes that they regulate.
TATA box- ( promoter)
TATA box (also called Goldberg-Hogness box) is a DNA sequence) is a
DNA sequence found in the promoter region) is a DNA sequence found in the
promoter region of genes in archaea) is a DNA sequence found in the
promoter region of genes in archaea and eukaryotes.
Considered to be the core promoter sequence, it is the binding site of either
general transcription factors , and is involved in the process of
transcriptionConsidered to be the core promoter sequence, it is the binding site
of either general transcription factors , and is involved in the process of
transcription by RNA polymerase .
The TATA box has the core DNA sequenceThe TATA box has the core DNA
sequence 5'-TATAAA-3' or a variant, which is usually followed by three or more
adenine bases.
It is usually located 25 base pairs upstream of the transcription site.
The sequence is believed to have remained consistent throughout much of the
evolutionaryThe sequence is believed to have remained consistent throughout
much of the evolutionary process, possibly originating in an ancient eukaryoticThe
sequence is believed to have remained consistent throughout much of the
evolutionary process, possibly originating in an ancient eukaryotic organism.
TATA BOX-
The TATA box is necessary for transcription because RNA polymersase II cannot
recognize the initiation sites on its own. The TATA box directs the RNA
polymersase II to the initiation site once the RNA polymerase has bound to the
TATA box. Yet another promblem occurs when the RNA polymerase II scans for
the TATA box. RNA polymerase II cannot recognize the TATA box on its own; it
has to use transcription factors to find the TATA box. After the transcription factors
bind to the TATA box, then RNA polymersase II can recognize and bind to the
TATA box
Prinbow box:
The Pribnow box or Pribnow-Schaller box is named after David Pribnow and
Heinz Schaller
it is the sequence TATAAT of six nucleotides of six nucleotides (thymine of six
nucleotides (thymine-adenine of six nucleotides (thymine-adenine-thymine of six
nucleotides (thymine-adenine-thymine-etc.) that is an essential part of a promoter
of six nucleotides (thymine-adenine-thymine-etc.) that is an essential part of a
promoter site on DNA of six nucleotides (thymine-adenine-thymine-etc.) that is an
essential part of a promoter site on DNA for transcription of six nucleotides
(thymine-adenine-thymine-etc.) that is an essential part of a promoter site on DNA
for transcription to occur in bacteria.
Pribnow box has a function similar to the TATA box has a function similar to the
TATA box that occurs in promoters in eukaryotes has a function similar to the
TATA box that occurs in promoters in eukaryotes and archaea: it is recognized and
Structural gene:
It’s a component of that strand of DNA which has 3’ to 5’ polarity ( as transcription
can occur only in 5’ to 3’ direction). This strand of DNA is called template strand or
master strand or antisense strand or ( _ ) strand.
The other strand which has a polarity of 5’ to 3’ is displaced during transcription.
This non template strand which does not take part in transcription is also called
sense or coding strand or plus ( + ) strand.
Monocistronic versus polycistronic mRNA
An mRNA molecule is said to be monocistronic when it contains the genetic
information to translatewhen it contains the genetic information to translate only a
single proteinwhen it contains the genetic information to translate only a single
protein. This is the case for most of the eukaryotic mRNAs.
polycistronic mRNA carries the information of several genes, which are translated
into several proteins. These proteins usually have a related function and are grouped
and regulated together in an operon.
Most of the mRNA found in bacteriaMost of the mRNA found in bacteria and
archea are polycistronic.
Dicistronic or bicistronic is the term used to describe an mRNA that encodes only
two proteins.
Specific part of the strand- operon:
In genetics, an operon is a functioning unit of genomic DNA containing a cluster
of genes under the control of a single regulatory signal or promoter.

Originally operons were thought to exist solely in prokaryotesOriginally operons

were thought to exist solely in prokaryotes, but since the discovery of the first
operons in eukaryotes in the early 1990s more evidence has arisen to suggest
they are more common than previously assumed.
Necessity of transcription:
In eukaryotes transcription generally occurs in G1 and G2 phase of cell
cycle inside the nucleus.
The transcription products move out for translation.
in prokaryotes transcription occurs in contact with the cytoplasm and as
their DNA lies in the cytoplasm.
Most imp. Necessity- DNA dependent RNA polymerase..
RNA POLYMERASE-
RNA polymerase (RNAP or RNApol) is an enzyme that produces
RNARNA. In cellsRNA. In cells, RNAP is needed for constructing RNA
chains from DNA genesRNA. In cells, RNAP is needed for constructing
RNA chains from DNA genes as templates, a process called transcription.
RNA polymerase enzymes are essential to life and are found in all
organisms and many virusesRNA polymerase enzymes are essential to life
and are found in all organisms and many viruses. In chemical terms, RNAP
is a nucleotidyl transferaseRNA polymerase enzymes are essential to life
and are found in all organisms and many viruses. In chemical terms, RNAP
is a nucleotidyl transferase that polymerizesRNA polymerase enzymes are
essential to life and are found in all organisms and many viruses. In
chemical terms, RNAP is a nucleotidyl transferase that polymerizes
ribonucleotidesRNA polymerase enzymes are essential to life and are
found in all organisms and many viruses. In chemical terms, RNAP is a
Prokaryotic RNAP:

Prokaryotes have only one RNA polymerase which synthesize all types of RNA.
They have polypeptide chains beta, beta’ alpha , alpha’ and sigma.
Sigma factor recognizes the start signal or promoter region of DNA.
The part of polymerase enzyme minus the sigma factor is called core enzyme.
Termination factor is rho.
Process of transcription-
1. activation of ribonucleotides- prior to transcription the nucleotides are
activated through phosphorylation.
Its done under the effect of phosphorylase.

Phosphorylases are enzymes that catalyze the addition of a phosphate group

from an inorganic phosphate (phosphate + hydrogen) to an acceptor.
DNA TEMPLATE-
On specific signals, segments of DNA corresponding to one or more cistrons
becomes ready to transcribe.
Cistron- A section of DNA that contains the genetic code for a single polypeptide
and functions as a hereditary unit.
Each such DNA transcription segment has a promoter region , initiation site,
coding region and a terminator region.
A promoter region has RNA polymerase binding site and recognition site.
Chain opening occurs region occupied in the TATA box.
Enzymes required for chain separation are unwindase and single stranded binding
proteins.
Terminator region has polyA or pallindromic sequence.
RNA polymerase (common in prokaryotes and specific in eukaryotes) binds itself
to the promoter region.
The two strands of DNA uncoil progressively from the site of polymerase
binding.
Pallindromes-
Base pairing-
Ribonucleotide triphosphates come to lie opposite the nitrogen bases of the
DNA template ( antisense strand).
A pyrophosphate is released from each ribonucleotide triphosphate to form
ribonucleotide.
the pyrophosphate is hydrolyzed with the help of enzyme pyrophosphatase.
It releases energy.
Chain formation- rnap joins.
With the help of RNA polymerase the adjacent
ribonucleotide held over DNA template join to form
RNA chain.
In prokaryotes a single polymerase recogonises the
promoter and initiation region.
In eukaryotes , there are separate transcription factors
and RNA polymerases for activation of transcription.
As the rna chain formation initiates , the sigma factor of
the RNA polymerase separates.
The core enzyme moves along the DNA template causing
elongation of RNA chain .
Rna polymerase moves along the DNA template causing elongation of RNA at the
rate of some 30 nucleotides per second.
RNA synthesis stops as soon as polymerase reaches the terminator region. Rho
factor is required for this.
Terminator region has a stop signal.
It also has 4-8 nucleotides.
Separation of RNA-
Termination or rho factor has ATPase activity. It helps in release of completed
RNA chain.
the released RNA is called primary transcript.
It is processed to form functional RNAs.
In many prokaryotes, the structural genes of related functions are generally
grouped together in operons.
An operon is transcribed as a single unit. Such a transcription unit is polycistonic
mRNA.
In eukaryotes, the transcription unit is a monocistronic mRNA .
Duplex formation:
After the release of primary transcript , the two strands of DNA establish
linkages amongst complementary base pairs.
Gyrases, unwindases and SSB proteins are released.
Consequently the double helical form of DNA is resumed.
Post transcriptional processing:
Primary transcript is often larger than the functional RNAs. This primary transcript
is called heterogenous nuclear RNA or hnRNA.
PTP is required to convert primary transcript of all types of RNA into functional
RNA. Its of four types-
Cleavage.
Splicing.
Terminal additions.
Nucleotide modifications.
Cleaving:

Larger RNA precursors are cleaved to form smaller RNA’s .

Primary transcript of rRNA is 45 S in eukaryotes.
It is cleaved by ribonuclease - p.
Splicing-
Eukaryotic transcripts possess extra segements called introns or intervening
sequences or non coding sequences.
They do not appear in mature or processed rna.
The functional coding sequence are called exons.
Splicing is removal of of introns and fusion of exons to form functional RNA.
This is needed for the typical eukaryoticThis is needed for the typical eukaryotic
messenger RNAThis is needed for the typical eukaryotic messenger RNA before
it can be used to produce a correct protein through translation.
Introns and exons-
For many eukaryotic introns, splicing is done in a series of reactions which are
catalyzedFor many eukaryotic introns, splicing is done in a series of reactions
which are catalyzed by the spliceosomeFor many eukaryotic introns, splicing is
done in a series of reactions which are catalyzed by the spliceosome, a complex of
small nuclear ribonucleoproteins (snRNPs),
but there are also self-splicing introns.
Each intron starts with dinucleotide GU and ends with dinucleotide AG. ( GU-
AG rule).
They are recognized by components of splicing apparatus. ( SnRNPs)
It has U1, U2, U4, U5, U6.
A complex called splicosome Is formed between 5’ ( GU) end and 3’ end (AG) of
intron.
Splicing is catalyzed by the spliceosome which is a large RNA-protein complex
composed of five small nuclear ribonucleoproteins (snRNPs, pronounced 'snurps'
). The RNA components of snRNPs interact with the intron and may be involved
in catalysis
Energy is obtained from ATP.
It removes the intron.
The adjacent exons are brought together.
The ends are sealed by RNA ligase.
Terminal addition-
Capping and tailing-additional nucleotides are added to the ends of RNAs for
specific functions.
Capping- Shortly after the start of transcription, the 5' end of the mRNA being
synthesized is bound by a cap-synthesizing complex associated with RNA
polymerase.
A 5' cap (also termed an RNA cap, an RNA 7-methylguanosine cap, or an RNA
m7G cap) is a modified guanine nucleotide that has been added to the "front" or
5' end of a eukaryotic messenger RNA shortly after the start of transcription.
The 5' cap consists of a terminal 7-methylguanosine residue
Its presence is critical for recognition by the ribosomeIts presence is critical for
recognition by the ribosome and protection from Rnases. Cap is formed by
modification of GTP into 7 methyl guanosine or 7mG.
polyadenylation -
Poly a segments are added at 3’ end of mrna.
200- 300 residues are added.
RNA: Ribonucleic acid
RNA-
It’s a single chain polyribonucleotide which functions as a carrier of coded
information or hereditary information from DNA to cytoplasm for taking part in
protein and enzyme synthesis.
At places RNA may appear double stranded due to folding or coiling of the double
strand.
It contains 70- 12000 ribonucleotides joined end to end.
Axis:
Types of RNA-

There are six types of RNA – ribosomal , transfer, messenger,

genetic(genomic), small nuclear and small cytoplasmic.
RNA is genomic in some viruses like TMV, HIV, H. Influenza virus.
It is double stranded in reoviruses, wound tumor viruse, rice dwarf virus,
mycophages.
Types of RNA – major.

• Messenger (mRNA) – single-stranded, complementary copy of

DNA

• Transfer RNA (tRNA) contains a binding site for mRNA codon

and a binding site for a specific amino acid

• Ribosomal RNA (rRNA) – component of the ribosome

Messenger RNA:

The term messenger RNA has been proposed by jacob and monod.
Its along RNA that constitutes 2- 5% of the total RNA content of the cell.
It brings instructions from the DNA for the formation of a particular type of
polypeptide.
Aka informational RNA.
The genetic code present in nucleotides is the instruction from DNA.
Three adjacent nitrogen bases specify a particular amino acid.
Formation of polypeptide occurs over the ribosome.
mRNA gets attached to the ribosome.
tRNA are induced to bring amino acids in a particular sequence according to
the sequence of codons present over the mRNA.
Methylation at 5’ terminus-
The 5' cap is a specially altered nucleotide is a specially altered nucleotide on the
5' end is a specially altered nucleotide on the 5' end of precursor messenger
RNA is a specially altered nucleotide on the 5' end of precursor messenger RNA
and some other primary RNA transcripts as found in eukaryotes is a specially
altered nucleotide on the 5' end of precursor messenger RNA and some other
primary RNA transcripts as found in eukaryotes. The process of 5' capping is vital
to creating mature is a specially altered nucleotide on the 5' end of precursor
messenger RNA and some other primary RNA transcripts as found in
eukaryotes. The process of 5' capping is vital to creating mature messenger RNA
is a specially altered nucleotide on the 5' end of precursor messenger RNA and
some other primary RNA transcripts as found in eukaryotes. The process of 5'
capping is vital to creating mature messenger RNA, which is then able to undergo
translation.
Capping ensures the messenger RNA's stability while it undergoes translation in
Polyadenylation-
Polyadenylation is the addition of a poly(A) tail to an RNA to an RNA
molecule. The poly(A) tail consists of multiple adenosine monophosphates to an
RNA molecule. The poly(A) tail consists of multiple adenosine monophosphates; in
other words, it is a stretch of RNA that has only adenine to an RNA molecule.
The poly(A) tail consists of multiple adenosine monophosphates; in other words, it
is a stretch of RNA that has only adenine bases. In eukaryotes to an RNA
molecule. The poly(A) tail consists of multiple adenosine monophosphates; in
other words, it is a stretch of RNA that has only adenine bases. In eukaryotes,
polyadenylation is part of the process that produces mature messenger RNA to an
RNA molecule. The poly(A) tail consists of multiple adenosine monophosphates; in
other words, it is a stretch of RNA that has only adenine bases. In eukaryotes,
polyadenylation is part of the process that produces mature messenger RNA
(mRNA) for translation to an RNA molecule. The poly(A) tail consists of multiple
Cap is followed by initaition codon either immediately or after a small non coding
leader region.
Then there is coding region followed by termination codon.
After termination code there is a small non coding trailer region and poly A area
at the 3’ terminus.
These leader and terminal region are called UTR region. ( untranslated regions).
An mrna may specify only a single polypeptide or a number of them. The former
called monoscistronic while latter is known as polycistronic.
Eukaryotes mra is usually monocistronic while prokaryotes have polycistronic
mrna.
1. Ribosomal Rna-
The term ribosomal rna has been proposed by kurland in 1960.
It accounts for most abundant ( 70 – 80 %) of total.
23 s , 28 s are the longest of all rna.
Ribosomal ribonucleic acid (rRNA) is the RNA) is the RNA component of the
ribosome) is the RNA component of the ribosome, the organelle) is the RNA
component of the ribosome, the organelle that is the site of protein synthesis in
all living cells) is the RNA component of the ribosome, the organelle that is the
site of protein synthesis in all living cells. Ribosomal RNA provides a mechanism
for decoding mRNA) is the RNA component of the ribosome, the organelle that is
the site of protein synthesis in all living cells. Ribosomal RNA provides a
mechanism for decoding mRNA into amino acids) is the RNA component of the
ribosome, the organelle that is the site of protein synthesis in all living cells.
Ribosomal RNA provides a mechanism for decoding mRNA into amino acids and
The ribosomal RNAs form two subunits, the large subunit (LSU) and small
subunit (SSU). mRNA is sandwiched between the small and large subunits and the
ribosome catalyzes the formation of a peptide bond between the 2 amino acids
that are contained in the tRNA.
Svedberg unit-
The Svedberg Unit (S)
As ribosomal particles were first isolated from cell lysates by ultracentrifugation,
the ribosomes and their sub-particles were named according to their
sedimentation characteristics during centrifugation.
The sedimentation properties of a particle depends on its molecular size and
geometrical shape.
The sedimentation characteristics also depend on the physical properties of the
solution through which the particle is sedimenting.
The two eukaryotic ribosomal subunits have sedimentation coefficients of 40 x 10-13
and 60 x 10-13. As one Svedberg (S) unit is 10-13, the two ribosomal subunits are
referred to as the 40S and the 60S ribosomal subunits.
Types of ribosomal rna-
Depending on their sedimentation coefficient , rna of eukaryotes are of 4 types-
28s,18s,5.8s and 5s.
Prokaryotic ribosome's have three types of RNA- 23s, 16s and 5s.
28s, 5.8s and 5s ( 23 s and 5s in prokaryotes ) occur in large subunit of ribosome.
While 18s ( 16s ) in prokaryotes are found in smaller subunit of ribosome.
Functions-
rRNA bind protein molecules and give rise to ribosomes.
Transfer rna-
It is also called soluble or s rna.
There are over 100 types of tRNA.
15% of total.
Smallest with 73 – 93 nucleotides and sedimentation coeff of 4S.
The nitrogen bases of several of its nucleotides get modified
Eg- pseudouridine ( $ ), dihydrouridine , ionosine , ribo-thymidine. (rt).
This causes coiling of otherwise single stranded tRNA into L shaped form.
Structure of tRNA
▪ small chain of about 80 nucleotides.
▪ transfers specific amino acid molecules to a
growing polypeptide chain.
▪ clover leaf model with 5 arms each with a
specific function.
▪ Also has an anticodon region that can base
pair with the codon region on the mRNA.
Three dimensional structure given by , klug(1974).
Two dimensional by holley ( 1965).
About half of the nucleotides are base paired to produce paired stems.
Five regions are unpaired or single stranded.
1. AA binding site.
2.T $ C loop.
3. DHU loop.
4. extra arm.
5.anticodon loop.
T- rna.
Parts of t rna.
Anticodon loop- it has 7 bases out of which three form anticodon ( nodoc) for
recogonising and attaching to the codon of M rna.
AA – Binding site – it is amino acid binding site. The site lies at 3’ end opposite the
anticodon and has CCA- 0H group.
The 5’ end bears G.
Amino acid and AA binding site and anticodon are the two recognition sites of
tRNA.
T$C loop- it has 7 bases out of which pseudouridine ($) and Rt( ribothymidine)
are unusual bases.. The loop is the site for attaching to the ribosome.
DHU loop- the loop contains 8 – 12 bases.
It is the largest loop and has dihydrouridine.
It is binding site for aminoacyl synthetase enzyme.
EXTRA ARM- it is a variable side arm or loop which lies between T$C loop
and anticodon. It Is not present in all tRNAs .
Functions:
Crick postulated that tRNA is adapter molecule.
It is meant for transferring amino acids to ribosomes for synthesis of
polypeptides.
An aminoacyl tRNA synthetase (aaRS) is an enzyme) is an enzyme that
catalyzes the esterification) is an enzyme that catalyzes the esterification of a
specific amino acid) is an enzyme that catalyzes the esterification of a
specific amino acid or its precursor to one of all its compatible cognate
tRNAs) is an enzyme that catalyzes the esterification of a specific amino acid
or its precursor to one of all its compatible cognate tRNAs to form an
aminoacyl-tRNA) is an enzyme that catalyzes the esterification of a specific
amino acid or its precursor to one of all its compatible cognate tRNAs to
form an aminoacyl-tRNA. This is sometimes called "charging" the tRNA
with the amino acid. Once the tRNA is charged, a ribosome) is an enzyme
Protein translation-
Machinery- it consists of ribosomes, amino acids, Mrna, T RNA, and aminoacyl
trna synthetase.
RIBOSOMES- protein synthesis occurs over the ribosomes. That’s why
ribosomes are called protein factories.
Each ribosome has two unequal parts, small and large.
The large subunit has a groove for pushing out the newly formed polypeptide
and protecting the same from cellular enzymes.
The smaller subunit fits over the larger one like a cap but leaves a tunnel for
mRNA .
Association-
The two subunits come together only during protein translation.
This phenomenon is called association.
Mg2+ is essential for it.
Soon after the completion of protein synthesis ,the subunits separate.
The phenomenon is called dissociation.
Different parts of ribosomes-
A tunnel for m rna . It lies in between the two subunits .
A groove for passage of newly synthesized polypeptide. This is part of the
larger subunit.
Reactive sites-
There are three reactive sites. –A ,P ( D) , and E.
A site ( amino acyl site or acceptor site ) is situated on the larger subunit of
ribosome. It faces the tunnel between the two subunits.
P- site (peptidyl transfer or donor site) is jointly contributed by the two ribosomal
subunits.
E site or exit site is part of the larger subunit facing the tunnel on the other side.
The ribosomeThe ribosome has three sites:
the A site, the P site, and the E site.
The A site is the point of entry for the aminoacyl tRNA (except for the
first aminoacyl tRNA, fMet-tRNAfMet, which enters at the P site).
The P site is where the peptidyl tRNA is formed in the ribosome.
And the E site which is the exit site of the now uncharged tRNA after it
gives its amino acid to the growing peptide chain.
tRNA (Transfer RNA)
tRNAs are encoded by tRNA genes.

All tRNA molecules are similar in size and shape.

All tRNAs have CCA at the 3' end to which the amino acid attaches.

At the other "end" of the tRNA molecule is the anticodon, which, during
translation, "reads" the matching codon on the mRNA.
T rna-
Aka transfer or soluble RNA.
Pick up a particual amino acid at 3’ end by the process called charging.
The charged T rna take the A.A to m rna over codons corresponded by
anticodon.
A T rna can pick up only a specific amino acid ,though an amino acid can be
specified by 2- 6 T rnas.
T rna comes in contact with ribosome at TC arm.
Also comes in contact with the enzyme amino acyl trna synthetase at DHU arm.
Adding of amino acid to T rna-
1. ACTIVATION OF A.A- its carried out by activating enzymes known as aminoacyl
t RNA synthetase ( zamecnik and hoagland).
In the presence of ATP , an amino acid combines with its specific amino acyl t rna
synthetase and subsequently to t RNA.
mg+ is required.
It produces an amino acid adenylate enzyme complex.
The energy made available to the amino acids is alter made use while forming
peptide bonds.
Hydrolysis of pyrophosphate with the help of enzymes pyrophosphatase provides
energy for driving the initial reactions.
Adding an Amino Acid to tRNA
An enzyme called aminoacyl-tRNA synthetase adds the correct amino
acid to its tRNA.
It’s the activating enzyme.
Charging or aminoacylation:
The correct amino acid is added to its tRNA by a specific enzyme called an
aminoacyl-tRNA synthetase.
The process is called aminoacylation, or charging.
Since there are 20 amino acids, there are 20 aminoacyl-tRNA synthetases.
All tRNAs with the same amino acid are charged by the same enzyme, even
though the tRNA sequences, including anticodons, differ.
Amino acids:
Hundreds of different types of proteins may be manufactured in a single
cell.
All types of proteins may be formed from the same amino acids.
It is the arrangement of amino acids in the polypeptides and the number of
them which provide specificity to the proteins.
Nearly 20 amino acids occur in the cellular pool.
M rna-
Brings out coded information from the DNA.
However the codons of mRNA are not recognized by amino acids but by
anticodons of their adapter molecules.
Initiation of translation-
It requires factors called initiation factors. There are three initiation factors in
prokaryotes- IF3, IF2, IF1.
Eukaryotes have nine initiation factors. eIF2., Eif1, eIF4A, e IF4B, eIF4C, eIF4D,
eIF5, eIF6.
Out of these IF3 or eIF2 is attached to smaller subunit of ribosome in the
dissociated state.
M rna attches itself to the smaller subunit of ribosome In the region of its cap.
the attachment is such that initaition codon of Mrna ( AUG, GUG) comes to lie
at the peptidyl site.
Initiation factors already present in the smaller subunit catalyses the reaction.
Mechanism-
Aminoacyl T rna complex specific for the initiation codon (methionine trna or
valine tna) reaches the P (d) site . Anticodon (UAC of trna for methi) establishes
temporary hydrogen bonds with the initiation codon.( AUG OF m RNA).
The codon anticodon reaction occurs in the presence of initiation factor eIF3 in
eukaryotes and IF2 in prokaryotes.
Energy is provided by GTP.
The initiating methionine accepting t rna is charged with non formylated
methionine in the cytoplasm of eukaryotes and formylated methionine in
prokaryotes , plastids and mitochondria.
Trna engaged in transferring formylated methionine is different than the one that
transfers nonformylated methionine.
In the presence of mg2+, the larger subunit of ribosome now combines with 40s
mrna – t rna complex to form intact ribosome.
it requires initiation factors IF1 in prokaryotes and factors eIF1, eIF4 in
eukaryotes.
The intact ribosome encloses the Mrna – T rna complex present at the p site and
keeps the A site exposed.
Elongation-( polypep chain formation)
Attack on A site-
An aminoacyl t rna complex reaches the A site and attaches to m rna codon next
to the initiation codon with the help of its anticodon.
This step requires GTP and an elongation factor.
E EF1 in eukaryotes and EF-TU and EF-Ts in prokaryotes.
Peptidyl transferase:
peptide bond- by peptidyl transferase
A peptide bond CO- NH is established between the carboxyl group( - COOH ) of
amino acid attached to t RNA at P site and amino group( - NH2) of amino acid
attached to t RNA at A site.
This reaction is catalysed by peptidyl transferase which is an rna enzyme.
The connection between the Trna at the P site and the A.A breaks off.
The free t rna of the p site slips into E site and from there to the outside with the
help of G factor
Translocation:
Soon after first peptide linkage and slipping of the freed tRNA of the P site, the
ribosome or mrna rotates slightly.
The process is called translocation, requires translocase.
( EF-G in prokaryotes, Eef2 in eukaryotes) and energy from GTP.
As a result of translocation the A site codon along with peptidyl t RNA complex
reaches the P site.
A new codon reaches the A site.
It attracts a new aminoacyl t rna complex.
The elongated peptide chain or polypeptide lies in the groove of the larger subunit
of ribosome to protect itself from cellular enzymes because it is prone to break
down due to its extended nature.
For every single amino acid incorporated in peptide chain one atp and two gtp are
used.
Termination:
Polypeptide synthesis is terminated when a nonsense codon of mrna reaches the
A site.
There are nonsense codons are not recogonised by any of the t rnas.
Therefore no aminoacyl t rna reaches the A site.
The p site t rna is hydrolysed and the completed polypeptide is released in the
presence of gtp dependent releasing factors.
It is eRF1 in eukaryotes and RF1 and RF2 in prokaryotes.
In prokaryotes RF1 is specific for UAG and UAA.
RF2 is specific for UAA , UGA.
Polysomes-
Polyribosomes (or polysomes) also known as ergosomes are a cluster of
ribosomesribosomes, bound to a mRNA molecule.
Many ribosomes read one mRNA simultaneously, progressing along the mRNA
to synthesize the same protein.
They may appear as clusters, linear polysomes, or circular rosettes on
microscopy, but mainly circular.
The 5' 7-methylguanosine capThe 5' 7-methylguanosine cap and 3' poly(A) tail
present on eukaryotic mRNA aid in this process
The adjacent ribosomes on polysomes are 340 angstorm or 34 nm apart.
RNA Polymerase (Prokaryotic)

• RNA polymerase (RNAP or RNApol) is an enzyme that makes a

RNA copy of a DNA or RNA template.
• RNA polymerase is a nucleotidyl transferase that polymerizes
ribonucleotides at the 3' end of an RNA transcript.
• Prokaryotic cells: only one type of RNA polymerase present
• The core enzyme has 5 subunits: 2 α, and one each of β, β’ and σ
• RNAP represented by α2ββ’σ (in addition ω)
• α2ββ’ form the core enzyme and σ factor is not firmly binded
• α2: responsible for promoter binding
• ββ’: form the catalytic center which helps in unwinding the DNA
molecule
• σ: selects initiation sites on the DNA
• ω: restores denatured RNA
RNA Polymerase (Eukaryotic)
Several types: characterized by the type of RNA they synthesize
More complex structures (each with 2 large subunits & 10-14 smaller subunits)
Types:
• RNA polymerase I: synthesizes a large (28S, 18S and 5.8S) rRNAs
• RNA polymerase II: synthesizes mRNAs and most snRNA and
microRNAs.
• RNA polymerase III: synthesizes tRNAs, 5S rRNA and other small RNAs
found in the nucleus and cytosol
RNA Polymerase Action
Occurs in the nucleus

One strand is used as a template to produce mRNA

Three main stages:

1. Initiation
2. Elongation
3. Termination
Transcription: Initiation
RNA polymerase binds to the promoter region
DNA unwinds
In E. coli, the most common type of promoter contains two
conserved sequences centered at -10 and – 35
In eukaryotes, promoter is located from approximately -40 to +50
relative to the start site
The most prominent (though not found in all promoters) is the
TATA box (5'-TATAAA-3') located at approximately -30
Transcription: Elongation

30–50 nucleotides/second
RNA nucleotides pair with DNA template A–U, T–A,
G–C, and C–G
Transcription: Termination

• RNA polymerase reaches the terminator and mRNA

(pre) is released
Product of Transcription: Pre-mRNA (Eukaryotes)
Large pre-mRNA molecules are composed of exons and introns
Exons are nucleotide sequences that are transcribed and translated
Introns are nucleotide sequences that are transcribed but not
translated
0–75 per gene
Size ranges from 100–100,000 nucleotides
Processing Pre-mRNA
Introns are removed
Exons are spliced together
Cap of 7 methyl guanosine or 7mg is added to the 5’ end
The cap helps attach mRNA to the ribosome
Protect 5’ end from exonucleolytic attack
Mark mRNAs as “self”
Poly-A tail is added to the 3’ end
interact with PolyA-binding protein: important for translation.
The mRNA is transported out of the nucleus to the ribosome for
translation
Splicing, capping and tailing
TRANSLATION
The sequence of bases in mRNA are copied into a sequence of
amino acids in a protein
(mRNA →protein)
occurs in cytoplasm on ribosomes
Requires various factors: enzymes, amino acids, energy, mRNA,
rRNA and tRNA
Three steps
Initiation
Elongation
Termination
 Steps in Translation: Initiation

1. Activation of amino acid with the help of ATP and amino acyl
synthetase enzyme
2. Attachment of the activated amino acid to the 3’ end of the
tRNA molecule
3. Small ribosomal subunit binds to mRNA
4. tRNA carrying methionine (f-met in case of prokaryotes) binds to
the start codon (AUG) within P site- Initiation of polypeptide
5. Forms initiation complex
Initiation
Initiation
 Steps in Translation: Elongation
1. Ribosome moves down mRNA in 5’ to 3’ direction
2. tRNA for the second amino acid binds to the mRNA within the second
ribosomal binding site (A site)
3. Peptide bonds forms between methionine and second amino acid with
the help of the enzyme peptidyl transferase
4. Peptide bond forms
5. tRNA brings in the third amino acid into the A site
6. Translocation of peptidyl tRNA from site A to P uses energy in the form
of GTP
Elongation
Elongation
Steps in Translation: Termination

1. Elongation continues until the ribosomes reach a stop codon

(UAG, UAA, UGA)
2. No tRNA binds
3. Synthesis is completed and mRNA, tRNA, are released from
the ribosome
4. Polypeptide is folded into 3-dimensional shape
Termination
Amino acids to Proteins
Structure of Proteins
Primary sequence - sequence of amino acids
in a protein.

Secondary structure - depends on primary sequence.

Beta sheets and alpha helices.
Can be usually predicted from sequence.

Tertiary structure - 3-D structure of how the protein

folds.
Cannot be always predicted.

Quaternary structure - multimers. Many subunits each

of which is a polypeptide.
Regulation of gene expression: OPERON MODELS

Gene regulation

▪ Virtually every cell in the body contains a complete set of genes;

but they are not all turned on in every tissue
▪ Each cell in the body expresses only a small subset of genes at any
time
▪ Gene regulation occurs at the level of transcription or production
of mRNA
OPERON
▪ Set of structural genes (cistrons) and associated control genes
▪ OPERON = Structural genes (cistrons) + regulator gene (i) +
promotor gene (p) + operator gene (o)
▪ Regulator gene: produces repressor molecules and controls the
activity of operator gene
▪ Promotor gene: actual site of start of transcription; RNA
polymerase binds here
▪ Operator gene: controls the activity of structural genes; present
between the promotor and first structural genes
Lac Operon

▪ In addition to amino acids, E. coli cells also metabolize sugars in

their environment
▪ Monod and Jacob looked at the ability of E. coli cells to digest the
sugar lactose
▪ In the presence of the sugar lactose, E. coli makes an enzyme
called beta galactosidase which breaks down the sugar lactose so
the E. coli can digest it for food
▪ LAC Z gene in E coli that codes for the enzyme beta
galactosidase
Trp Operon

▪ In addition to sugars like glucose and lactose E. coli cells also

require amino acids
▪ One essential amino acid is tryptophan.
▪ When E. coli is swimming in tryptophan it will absorb the amino
acids from the media
▪ When tryptophan is not present in the media then the cell must
manufacture its’ own amino acids
▪ E. coli uses several proteins encoded by a cluster of 5 genes to
manufacture the amino acid tryptophan
▪ All 5 genes are transcribed together as a unit called an operon,
which produces a single long piece of mRNA for all the genes
▪ RNA polymerase binds to a promoter located at the beginning
of the first gene and proceeds down the DNA transcribing the
genes in sequence
 Madeejee
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