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▪ 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
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
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:
1. A DNA template
3. DNA polymerase
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)
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
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:
3′ 5′
5′ 3′
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
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
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
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
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′
5′
3′
3′
5′
growing 3′ telomerase
5′ replication fork
5′
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.
• 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?????
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.
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 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)
30–50 nucleotides/second
RNA nucleotides pair with DNA template A–U, T–A,
G–C, and C–G
Transcription: Termination
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
Gene regulation
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