Molecular biochemistry

STEP TO PG-MD/MS - DR.AKIF A.B
- DR.AKIF A.B

-Made up of monomer units called Nucleotides.
-Nucleotides are linked to each other by 3’-5’ Phosphodiester bond
-Backbone of nucleic acid = Sugar + Phosphate
NUCLEOTIDES
1) Nitrogen Bases : Purines
and pyramidines
2) Pentose sugar
3) Phosphate group
Nucleoside

-C1 of Pentose Sugar is linked to N9 of Purines or N1 of Pyramidines
-Most nucleotides are 5’ Nucleotides
-Base sequence og Nucleic Acid is written in 5’-3’ direction

1) At the physiological PH, the DNA molecules are:
A Positively charged.
B Negatively charged
C Neutral.
D Amphipathic

B) Negatively charged
DNA is negatively charged because of phosphate group

2) Nucleoside is made up of (PGI)
A. Pyramidine
B. Histone
C. Purine
D. Sugar
E. Phosphate

C. Purine
D. Sugar
A. Pyramidine
NUCLEOTIDES
1) Nitrogen Bases : Purines
and pyramidines
2) Pentose sugar
3) Phosphate group
Nucleoside
Ans.

3. Apart from occuring in Nucleic acids, Pyramidines are also present in
A. Theophylline
B. Theobromine
C. Flavin mononucleotide
D. Thiamine

D. Thiamine
Purine ring is present in Pyramidine ring is
present in
Theobromine Thiamine
Theophylline
Flavin Mononucleotide

3. Which one of the following procedures as routine technique for karyotyping
using light microscopy:
A C-banding
B G-banding
C Q-banding
D Brd V-standing

Ans. B) G-Banding
a. “The most commonly employed staining method uses a Giemsa stain and hence
is called G banding”
b. A Karyotype is a standard arrangement of a photographed or image stained
chromosomes, where chromosomes are in metaphase stage.
c. Mitosis is arrested in dividing cells in metaphase stage by use of colchicine.
d. In metaphase stage individual chromosomes take the form of two chromatids
connected at the centromeres.
i. Chromosomes are arranged in pairs
ii. Chromosome pairs arranged in decreasing order of length.

Staining allows identification of each individuals chromosome on the basis
of distinctive and reliable pattern of alternating light and dark bonds. One
of the following banding technique may be used.
STAINING OF CHROMOSOME
G—Banding Q— Banding C-Banding R-Banding
Giemsa bonding Quinacrine-
banding
Constitutive-
banding
Reverse staining
Giemsa banding
Most commonly
used
Demonstrates
bands
along
chromosome
Demonstrate
constitutive
hetero chromatin
Gives pattern
opposite to G-
banding

4. Which of the following is not a nitrogenous base
A. Adenine
B. Guanosine
C. Cytosine
D. Thymine

B. Guanosine
-Guanosine is a nucleoside
-Remaining are nitrogenous bases
-Guanine is a nitrogen base

5. On complete hydrolysis of DNA we will get all the following except
A Deoxy pentose sugar
B Phosphoric acid
C Adenosine
D Purine bases

C) Adenosine
- Nucleoside = Deoxy - Adenosine or adenosine = Base (Adenine) + sugar
- Adenosine = ribose + Adenine
-Deoxyadenosine = Deoxyribose + Adenine
Therefore Adenosine is present in RNA, not present DNA.

-DNA is a Polymer of deoxyribonucleotides
i. Adenine deoxyribonucleotide
ii. Thymine deoxyribonucleotide
iii. Guanine deoxyribonucleotide
iv. Cytosine deoxyribonucleotide
- Nucleotides joint by covalent 3’-5’ phosphodiester linkage
-Right handed Double helix structure of DNA is given by Waston and
Crick
-A combine with T (A = T) by two H2 bond C combines with G(C =G) by three
H2 bond
-Chargaff’s Rule — Ratio of purine (G+A) to pyrimidine (T+C) bases in the
DNA is always around 1.
- i.e. G + A / T + C = 1
-Antiparallel i.e one strand in 5’-3’ direction and other in 3’-5’ direction
-

- 6 types of DNA : A to E and Z
- most common TYPE of DNA is B-DNA
B-DNA
-10 base pair per turn
-Most stable
-This was actually explained by watson and
crick
-Length of one turn = 34 A* or 3.4nm
-Diameter oe width = 20 A* or 20nm
-Right handed helix ( Z-DNA is left handed)

TEMPLATE STRAND CODING STRAND
Non coding strand
Antisense strand Sense strand
-ve strand +ve strand
3’-5’ direction 5’-3’ direction
This strand is copied during m-
RNA synthesis

6. Thermostability of DNA is provided by
A G-C bonds
B A-T paining
C N-glycosolic bond
D Antiparallel arrangement

Ans. A G-C Bonds
- ‘A’ combines with ‘T’ by 2 hydrogen bonds i.e A = T while ‘C’ combines with
‘G’ by 3-Hydrogen bonds
i.e. C = G
Since it has 3 bonds it is responsible for its stability

Melting (Denaturation) of DNA
- Separation of 2 strands of DNA by breaking of Hydrogen Bonds
- DNA separated into two components strands either by:
1) increasing temperature or
2) decreasing salt concentration.
- Phosphodiester bond is not broken
- Primary structure is not disrupted, only secondary and tertiary structure
is disrupted.
- After denaturation (melting), of DNA, there is an increase in the optical
absorbance of purine and pyrimidine bases à a phenomenon referred as
Hyperchromicity of denaturation.
- DNA rich in G=C melt at higher temperature than that rich in A=T pairs.
- Formamide is used commonly in recombinant DNA experiments, Lowers the
‘Tm’ by destabilizing H bonds.

Melting (Denaturation) of DNA
-Melting Temperature (Tm) = Temperature at which half of strand is denatured
-Normal Tm = 85-95*C
-Tm = 2*no. of A=T Pairs + 3 * no. of C=G pairs

-3rd strand binds at major groove of B-DNA by Hydrogen bonding
-k/a Hoogsten Pairs

-High in Guanine content
- G –Quarlet DNA

- Regulates overwinding or underwining of DNA.
Topoisomerase I Topoisomerase II
Makes Single Stranded Nick In
DNA
Makes double stranded Nick
Removes –ve supercoils Removes –ve supercoils
Cant insert supercoils Can insert supercoils
ATP is not requires ATP is required
E.g.: Helicase DNA gyrase

Bacterial Topoisomerase Human topoisomerase
Ciprofloxacin Etoposide
Nalidixic acid Adriamycin
Daunorubocin

-DNA + Histone Protein
-Most abundant chromatin protein = Histone protein
-5 Types :
H1 Lysine rich
Linker protein
H2A,
H2B
Arginine rich
H3 Lysine rich
H4
-Beads of String appearance
-Nucleosomes are linked to each
other by 30bp k/a Linker.
- Basic protein

EUCHROMATIN HETEROCHROMATIN
Regions in DNA which is
transcriptionally active.
transcriptionally inactive
Chromatin is Less densely packed Densely packed
Stains less densely Stains densely

Constitutive Facultative
Always condensed At times condensed but other times it is
actively transcribed and thus
uncondensed and appears as
euchromatin
-Inactive
-Seen in centromere and ends of
telomere
X- chromosome
One X CHROMOSOME is inactive in
females (barr body) but at time of
embryogenesis it becomes active

-Y chromosome is Acrocentric
-X chromosome is submetacentric
-Telocentric chromosome is not seen in humans
-MC chromosome = Submetacentric

-It is increase of absorbance
-Measured by absorbance at 260nm
-It occurs when DNA is denatured
-ssDNA is more Hyperchromatic than dsDNA.

-Occurs in S phase of cell cycle
-Each DNA strands separates and acts as template strand on which
complementary strand is synthesised
-Base pairing rule is obeyed
-Semiconservative nature
-New strand is synthesised in 5’-3’ direction
-Synthesis of DNA in both strands is not similar :
-Leading strand : DNA is continuosly polymerised
-Lagging strand : DNA is discontinuosly polymerised (semi
discontinuous)

-Replication proceeds from multiple origin in each chromosomes in
eukaryotes including humans
-Replication obeys polarity
-Replication occurs in both directions along all of chromosome
-Both strands are replicated simultaneously
-Replication process generates ‘replication bubbles’

Common features between
prokaryotic and eukaryotic DNA
replication:
1) Semiconservative
2) Bidirectional
3) Semi discontinuous

TYPES FUNCTION
DNA Polymerase Alpha Primase
DNA Polymerae Beta DNA repair
DNAP gamma Mitochondrial DNA synthesis
DNAP delta Lagging strand synthesis
DNAP epsilon Leading strand synthesis

Polymerase I Gap filling following DNA
replication, repair and
recombination
Polymerase II Proof reading and repair
Polymerase III Leading strand synthesis,
Okazaki fragment synthesis

PROTEINS FUNCTION
DNA polymerases Deoxynucleotide polymerization
Helicases Unwinding of DNA
Topoisomerase Relieves torsional strain that
results from helicase induced
unwinding
DNA Primase Initiates synthesis of RNA primers
Single stranded binding proteins Prevents premature reannealing of
dsDNA
DNA ligase Seals the broken ends between
nascent chain and okazaki
fragments

-Telomeres are present at the end of
eukaryotic chromosome
-Telomeres consist of TG repeats
-During each replication telomere
shortens and thus cell dies later
-Early shortening of telomere is associated
with early aging and malignancy
-Telomerase lengthens telomere and it
is RNA dependent DNA
polymerase(Reverse Transcriptase)

Q. Highly repetitive DNA is seen in (PGI)
A. Cloning of DNA
B. Microsatellite DNA
C. Telomere
D. Centromere

Ans. C. Telomere
D. Centromere
-In human DNA around 30% of genome consists of repeatitive sequence
-The sequence are clustered in centromere and telomere
-They are transcriptionally inactive
-They mostly have structural role in chromosome.

An enzyme called Helicase breaks the
hydrogen bonds between the bases of
the two antiparallel strands.
The strands are initially split apart in
areas that are rich in A-T base pairs
forming a replication fork.
DNA Gyrase (also called
Topoisomerase) relieves tension that
builds up as a result of unwinding.
Single strand binding proteins
(SSBs) help to stabilise the single
stranded DNA.

RNA polymerase (also
known as RNA Primase)
synthesizes short RNA
nucleotides sequences that act
as primers (starters).
These essentially provide a
starting point for DNA
replication.

DNA Polymerase III can now start
synthesising the new DNA strand
using free DNA nucleotides.
However, DNA polymerase can only
read the original template (parent
strand) in the 3’ → 5’ direction
(making DNA 5’ → 3’).
This is not a problem on the leading
strand, because the DNA
polymerase can simply continue to
read along as the two parent stands
continue to unzip.

On the lagging strand DNA
polymerase moves away from the
replication fork.
As the strands continue to unzip more
DNA is exposed and new RNA
primers must be added.
As a result the lagging strand is
synthesised in short bursts as DNA
polymerase synthesizes DNA in-
between each of the RNA primers.

The newly synthesised lagging strand
now consists of both RNA and DNA
fragments.
The DNA fragments are known as
Okazaki fragments, after a Japanese
scientist who noticed that heating DNA
during replication, which separates the
strands, gave many small fragments of
DNA.
From this he concluded that one stand
must be synthesized in short bursts of
DNA.

DNA Polymerase I now removes the RNA
primers and replaces them with DNA

DNA Ligase joins the DNA fragments of
the lagging strand together to form one
continuous length of DNA.

DNA
damaging
agents
Defects in
DNA
Repair
mechanism
Disorder
associated
UV lights
Chemicals
Pyrimidine
dimers
Nucleotide
excision
repair
Xeroderma
pigmentosa
Replication
errors
Mismatch
repair
HNPCC
Ionising
radiation
Homologous
recombination
Ataxia
telangiectasia

Key points:
Transcription is the process in which a gene's DNA sequence is copied
(transcribed) to make an RNA molecule.
RNA polymerase is the main transcription enzyme.
Transcription begins when RNA polymerase binds to a promoter sequence
near the beginning of a gene (directly or through helper proteins).
RNA polymerase uses one of the DNA strands (the template strand) as a
template to make a new, complementary RNA molecule.
Transcription ends in a process called termination. Termination depends on
sequences in the RNA, which signal that the transcript is finished.

(a) To initiate the transcription process, RNA polymerase, shown as a
large green blob, binds to a promoter sequence shown in dark
green on a double-stranded DNA molecule.
(b) Once bound, RNA polymerase and its associated proteins bend the DNA
to separate the two strands.
(c) A DNA sequence downstream of the promoter region is labeled the
termination site, and it indicates where the transcription process will
end.

RNA POLYMERASE DNA POLYMERASE
No Primer is needed Primer is needed
No proof reading activity proof reading activity
TYPE OF RNA
POLYMERASE
MAJOR PRODUCTS
RNA Polymerase I rRNA
RNA Polymerase II mRNA, miRNA,
SnRNA
RNA Polymerase III tRNA, 5s rRNA
-In Prokaryotes
there is only one type
of RNA Polymerase
-Eukaryotes :3
Types

Bacterial
Promoters
Eukaryotic
promoters
1) TATA Box 10 bp upstream of
start site of
Transcription
1) Golberg
Hogness Box
-Similar to TATA
Box
-25-35 bp
upstream
2) TGG Box 35 bp upstream 2) CAAT Box 70-80 bp
upstream

-Usually Promoters are located upstream of start site
of Transcription
- But Promoters for RNA Polymerase III is located
Downstream
-Promoters lie on the coding strand of DNA but
not on Template strand

(b) During the elongation phase, RNA polymerase adds nucleotides to a
growing mRNA chain.
The nucleotides of the mRNA are represented here as pink T-shaped
molecules; a curved red arrow indicates that they are added to the three-
prime end of the growing mRNA transcript.
Blue arrows show that the DNA is wound back up at the left side and is
unwound at the right side as RNA polymerase moves along the template
strand from left to right, as indicated by a green arrow.

TEMPLATE STRAND CODING STRAND
Non coding strand
Antisense strand Sense strand
-ve strand +ve strand
3’-5’ direction or 5’-3’ direction 5’-3’ direction or 3’-5’ direction
This strand is copied during m-
RNA synthesis

(c) When RNA polymerase reaches the termination site on the template
DNA strand, the mRNA transcript is separated from the DNA.
Here, the synthesized mRNA is shown as a separate molecule below the double-
stranded DNA, and a red arrow above the double-stranded DNA shows the
release of the RNA polymerase from the DNA.

- Primarily occurs at nucleus
1) 7-methylguanosine capping at 5’ end
3) Removal of Introns and Joining of
Exons called splicing
2) Addition of Poly A tail at 3’ end

RNA DNA
Mostly seen in cytoplasm Nucleus
Destroyed by alkali Not destroyed
Single stranded Double stranded

1) Transfer RNA (tRNA)
2)Ribosomal RNA (rRNA)
3) mi RNA
4) Si RNA

-Most abundant RNA
-40S Ribosome = 18 SrRNA + 30 Proteins
-60S Ribosome = 5S rRNA + 5.8 + 28S
-Synthesised in Nucleolus
-Some rRNA acts as Ribozymes

-Transfer RNA
-Transfers Amino acids from Cytoplasm to Ribosomes
Acceptor arm -Amino acid binding
site
- CCA at 3’end
Anticodon arm Binds with mRNA
D-ARM Dihydrouridine arm
- Detects aminoacyl
tRNA Synthase
ARM - Ribothymidine and
Pseudouridine

1) Dihydrouridine = One of double bond is decreased
2) Pseudouridine : Ribose and N2 base is linked by C-
C bond instead of C-N bond
3) Ribothymidine : methylation of one of uracil to
form Thymine
- Only RNA to contain Thymine is : Ribothymidine tRNA
4) Inosine : Contain Hypoxanthine

1) Reverse Transcriptase : RNA dependent DNA Polymerase
2) DNA Polymerase : DNA dependent DNA Polymerase
3) Primase: DNA dependent RNA Polymerase
4) RNA Polymerase : DNA dependent RNA Polymerase

Replication Transcription
Deoxyribonucleotides are added Ribonucleotides are added
Adenine pairs with Thymine Adenine pairs with Uracil
Both strands of DNA acts as
Template
One strand act as template and
other as Coding strand
A Primer is involved as DNA
polymerase cannot initiate DNA
synthesis on its own
Primer is not required
DNA dependent DNA Polymerase
is the enzyme
DNA dependet RNA polymerase is
the enzyme

-Triplet nucleotide sequence present in mRNA representing specific Amino Acid.
-If 1 base represents 1 amino acid = then Total 4 Amino Acids (4 )
-If 2 base represents 1 amino acids = 4 = 16 amino acids
-If 3 base = 4 = 64 Amino Acids
-If 4 base = 4 = 256
1
2
3
4

EUKARYOTIC PROKARYOTIC
mRNA is monocistronic Polycistronic
Translation occurs only once
transcription is complete
Translation can occur even
before transcription is
complete
Initiating Amino Acid =
Methionione
N-Formyl Methionine
- mRNA is always translated from 5’ to 3’ direction.

-Specific Amino acid is attached to acceptor arm(3’)
of t-RNA by Amino-acyl-t-RNA synthase
-2 ATPs are used
-Amino acyl t-RNA synthase is specific for a Amino
acid and t-RNA
-Responsible for high fidelity of Translation of
Genetic message

There is no t-RNA for Hydroxylysine
and Hydroxyproline since they are
formed by post translation
modification of Lysine and Proline

80S Ribosome = 60S + 40 S
-2 sites for t-RNA on Ribosomes = P site( Peptidyl) + A site (Amino acyl)
-Initially t-RNA bind to P-site

EIF2 + GTP
EIF2 - GTP
EIF2 –GTP-t-
RNA
43 S
preinitiation
complex Binds to AUG
on m-RNA at
5’ end
48S initiation
complex
Binds with
60S
ribosomes
Forms 80S
initiation
complex
Release of all
elongation
factors(Eif2-
GTP)
Methionine-t-RNA
Binds to 40S ribosomes
T-RNA is at P
site

SHINE –DALGARNO SEQUENCE
-In Prokaryotes
- Codon sequence near initiator codon which facilitates binding of
Pre-Initiator complex with m-RNA
KOZAK-COSENSUS SEQUENCE
-In Eukaryotes
- Codon sequence near initiator codon which facilitates binding of
Pre-Initiator complex with m-RNA

-New amino acid binds with t-RNA and then this forms complex with GTP
to attach to A site of Ribosome on m-RNA

There are three termination codons that are employed at the end of a
protein-coding sequence in mRNA: UAA, UAG, and UGA.
No tRNAs recognize these codons.
Thus, in the place of these tRNAs, one of several proteins, called release
factors, binds and facilitates release of the mRNA from the ribosome and
subsequent dissociation of the ribosome.

-AUG
-In Eukaryotes = AUG codes for Methionine
-In prokaryotes = AUG codes for N-Formyl-Methionine

Charging of t-RNA 2 Phosphates
Formation of 48 S Pre initiation
complex
1 ATP
Formation of 80 S initiation
complex
1 GTP
Binding of fresh Aminoacyl t-
RNA @ A- SITE
1 GTP
During Translocation 1 GTP
During Termination 1 GTP

UAG UGA UAA
Amber Opal Ochre
EXCEPTIONS
UAG can be recoded to Pyrollysine
UGA can be recoded to Selenocysteine
UGA also codes for Tryptophan in Mitochondrial DNA

1) Trimming
2) Glycosylation -N-Glycosylation occurs in Endoplasmic
Reticulum
-O-Glycosylation occurs in Golgi Apparatus
3) Hydroxylation Of proline and Lysine gives Hydroxyproline
and Hydroxy Lysine
4) Gamma
Carboxylation
Of Vit. K dependent clotting factors ( 2,7,9,10)
5) Protein folding Chaperons
6) Protein degradation Ubiquitin

Molecular biochemistry

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Molecular biochemistry