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Biochemistry JSS Medical College: DR - Prashant Vishwanath
Biochemistry JSS Medical College: DR - Prashant Vishwanath
Biology
&
Genetics
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Learning Objectives
▪ Central dogma of molecular biology
▪ Structure of DNA
▪ Explain prokaryotic DNA Replication
▪ Differences between prokaryotic and
eukaryotic DNA replication
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
The Central Dogma
▪ This order of events is called the central
dogma of molecular biology:
DNA RNA P R T E N
O I
Transcription
Replication Translation
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Structure of DNA
▪ DNA is a polydeoxyribonucleotide
▪ 3’ – 5’ Phosphodiester Linkages
▪ Double stranded molecule
▪ Two strands wind around each other to
form a double helix
▪ In eukaryotes it is found associated with
nucleoproteins
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
DNA Replication
▪ Semi Conservative
▪ Slightly different in
eukaryotes and
prokaryotes
▪ Initiation of Replication
commits the cell to
continue the process till
the entire genome has
been replicated
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Steps of DNA Replication
▪ Initiation
▪ Synthesis of RNA Primer
▪ Elongation of DNA strand
▪ Proof reading of synthesized strands
▪ Removal of RNA primers
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Separation of the two
complementary DNA strands
▪ DNA replication begins at a single unique
nucleotide sequence – origin of replication
▪ In eukaryotes, replication begins at
multiple sites along the DNA helix
▪ These sites usually have a short sequence
composed exclusively of AT base pairs :
“Consensus Sequence”
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Formation of the Replication
Fork
▪ Two strands unwind
and form a “V”
▪ Replication Fork
moves along the
DNA molecule as
synthesis occurs
▪ Replication is
bidirectional
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Proteins Required for DNA
Strand Separation
▪ DnaA Protein – 20 to 50 monomers of
dnaA protein bind to specific nucleotide
sequences at the origin of replication,
which is particularly rich in AT base
pairs
▪ Single stranded DNA binding (SSB)
proteins – These bind only to single
stranded DNA. They bind cooperatively
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
▪ DNA Helicases – these enzymes bind
to single stranded DNA near replication
fork
▪ Unzip the double stranded DNA
▪ Requires energy provided by ATP
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
▪ DNA helicase separates the two DNA strands by
breaking the hydrogen bonds between them
▪ This generates positive supercoiling ahead of
each replication fork
– DNA topoisomerases travels ahead of the helicase and
▪ Type I & II
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Solving the problem of
Supercoils
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Type I DNA topoisomerase
▪ Reversibly cut one
strand of the double
helix
▪ Have nuclease and
ligase activities
▪ Do not require ATP
▪ Relax negative
supercoil in E coli, and
both positive and
negative supercoils in
eukaryotic cells
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Type II DNA topoisomerase
▪ Binds to DNA double helix
and make transient breaks
in both strands
▪ ATP requiring process
▪ Both positive and negative
supercoil can be relieved
▪ Anticancer agents,
etoposide, target human
topoisomerase II
▪ Ciprofloxacin targets
bacterial DNA gyrase
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Direction of DNA
Replication
▪ DNA polymerases are able to read only
3’ – 5’ direction and synthesize new
strands in 5’ – 3’ direction
▪ Therefore the synthesis of chains must
occur in opposite directions.
▪ It is different in both the strands
▪ Leading Strand & Lagging Strand
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Figure 11.13 “Three Dimensional” view of Replication Fork
Direction of synthesis
of leading strand
Direction of synthesis
Of lagging strand
Dr.Prashant Vishwanath
Biochemistry
Direction of fork movement
JSS Medical College
Lagging Strand
▪ The strand that is being copied away
from the replication fork is synthesized
discontinuously with small fragments of
DNA being copied
▪ These short stretches are called as
Okazaki fragments and are eventually
joined to become single continuous
strand
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
RNA Primers
▪ Short double stranded
region consisting of RNA
base paired to DNA
template with free hydroxyl
group on the 3’ end of RNA
strand
▪ This hydroxyl group serves
as acceptor for the 1st
nucleotide by action of DNA
polymerase
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Primase
▪ A specific RNA polymerase that synthesizes the short
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Chain Elongation
▪ Prokaryotic and eukaryotic DNA
polymerases elongate a new DNA by
adding deoxyribonuleotides, one at a
time.
▪ The sequence of addition is dictated by
the base sequence of template strand
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
DNA Polymerase III
▪ DNA Chain elongation is catalyzed by
DNA Polymerase III.
▪ All four deoxyribonucleotides must be
present for DNA elongation
▪ Pyrophosphate is released when each
nucleotide is added to growing chain
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Proofreading of Newly
Synthesized DNA
▪ DNA Polymerase III has in addition a
proofreading activity
▪ 3’ – 5’ exonuclease activity removes the
misplaced nucleotide
▪ 5’ – 3’ polymerase then replaces it with
the correct nucleotide
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Excision of RNA primer and their
replacement by DNA
▪ DNA polymerase III continues to synthesize
DNA on the lagging strand untill it is blocked
by proximity to an RNA primer
▪ The RNA is excised and the gap filled by DNA
polymerase I
▪ It has 5’-3’ exonuclease activity
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
DNA Ligase
▪ The final phosphodiester linkage between the
5’ phosphate group on the DNA chain
synthesized by DNA polymerase III and the 3’
hydroxyl group on the chain made by DNA
polymerase I is catalysed by DNA ligase
▪ This required energy which is provided by the
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Protein Synthesis
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Protein Synthesis
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Ribonucleic Acids (RNA)
▪ The job of RNA (ribonucleic acid) is to carry
messages from the DNA (in the nucleus) to
the ribosomes (in the cytoplasm).
▪ There are three types of RNA:
1. mRNA – carries a message from the
DNA to the cytoplasm
2. tRNA – transports amino acids to the
mRNA to make a protein
3. rRNA – make up ribosomes, which make
protein.
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Ribonucleic Acids (RNA)
double-stranded
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Ribonucleic Acids (RNA)
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Protein Synthesis
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Step 1: Transcription
– Initiation
– Elongation
– Termination
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Initiation
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Elongation
▪ RNA is synthesized from 5’ end to 3’ end
antiparallel to the DNA template
▪ RNA polymerase utilizes ribonucleotide
triphosphates (ATP, GTP, CTP and UTP) for
the formation of RNA.
▪ The sequence of nucleotide bases in the
mRNA is complementary to the template
DNA strand
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Termination
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Step One: Transcription
TACGCATGACTAGCAAGTCTAACT
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Step One: Transcription
TACGCATGACTAGCAAGTCTAACT
AUGCGUACUGAUCGUUCAGAUUGA
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
RNA Editing
▪ An mRNA molecule has to be “edited” in order to
be useful. There’s a lot of unnecessary
information that needs to be removed.
▪ An mRNA sequence that does NOT code for
protein is called an interon. A sequence that is
useful in making a protein is called an exon.
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
RNA Editing
DNA
transcriptio
pre-RNA (in n
nucleus) intero
exon 1 interon exon 2 n exon 3
RNA
interon
editing
intero
n
RNA (in
cytoplasm)
exon 1 exon 2 exon 3
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Step Two: Translation
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Step Two: Translation
▪ Problem:
– There are 20 different amino acids.
– There are 4 RNA bases.
A T C G
phe ile val pro ala his asn asp cys arg
leu met ser thr tyr gln lys glu trp gly
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Genetic code
▪ The three nucleotide base sequences in mRNA that act as
code words for amino acids in protein constitute the
genetic code
▪ The codons are composed of the four nucleotide bases
▪ These four bases produce 64 different combination
▪ Sixty one codons code for the 20 amino acids found in
protein
▪ The three codons UAA, UAG and UGA do not code for
amino acids
▪ They act as stop signals in protein synthesis
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
The Genetic Code
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Protein synthesis
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Requirement of the
components
▪ The protein synthesis may be considered as a
biochemical factory operating on the ribosomes
and requires many components
– Amino acids
– Ribosomes
– mRNA
– tRNA
– Energy sources – Both GTP and ATP
– Protein factors
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Activation of amino acids
▪ Amino acids are activated and attached to
tRNAs
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Initiation
▪ The initiation factors – IF1 and IF3 – first bind
with the free 30S subunit
▪ This is followed by the attachment of mRNA with
30S subunit
▪ Next, IF2 and methionine tRNA, in the presence
of GTP, bind with 30S ribosome
▪ The 50S ribosome associates with 30S to form
70S initiation complex and the initiation factors
are released
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Initiation codon
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Elongation
▪ Ribosomes elongate the polypeptide chain by a
sequential addition of amino acids to the growing
carboxyl end
▪ During elongation, the ribosome moves from 5’
end to 3’ end of the mRNA
▪ Elongation factors – EF-Tu and EF-Ts – in
association with GTP, facilitate elongation
▪ At the beginning, the initiation tRNA occupies the
‘P’ site of the ribosomes
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Elongation
▪ The incoming next amino acid corresponding to
codon on mRNA is deposited at ‘A’ site of the
ribosome
▪ The enzyme peptidyltransferase catalyses the
formation of peptide bond
▪ As the peptide bond formation occurs, the ribosome
moves to the next codon in the mRNA
▪ This process, called translocation, basically involves
the movement of growing peptide chain from A site
to P site of the ribisome
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Elongation
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Termination
▪ One of the three codons UAA, UAG and UGA act
as stop signals and terminate the growing
polypeptide
▪ These signal codons do not have specific tRNAs
to bind with them
▪ As the termination codon occupies the ribosomal
A site, the release factors – RF-1, RF-2 and RF-3
– bind with the codon
▪ These factors cause the hydrolytic breakdown of
the peptidyl-tRNA and release the newly
synthesized protein
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Post-translational
modifications
▪ The proteins synthesized in translation are, as
such, not functional
▪ Many changes take place in the polypeptides after
the protein synthesis is completed
▪ These modifications include trimming by
proteolytic degradation and covalent changes
which are collectively known as post-translational
modifications
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Inhibitors of protein
synthesis
▪ Translation is a complex process and is inhibited by
antibiotics
▪ Antibiotics are the substances produced by bacteria
or fungi which inhibit the growth of other organisms
▪ They interfere with the bacterial protein synthesis
and are harmless to higher organisms
▪ This is due to the fact that the process of translation
sufficiently differs between prokaryotes and
eukaryotes
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Examples
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
▪ Streptomycin and aminoglycoside are
bactericidal. Bind to 30S subunit of bacterial
ribosomes. Cause misreading of mRNA and
at high concentration completely inhibit the
initiation complex formation
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College
Inhibitors of DNA Replication
Drug Action (Inhibition of)
Antibacterial agents
Ciprofloxacin Bacterial DNA gyrase
Nalidixic Acid Bacterial DNA gyrase
Novobiocin Bacterial DNA gyrase
Anticancer Drugs
Etoposide Human topoisomerase
Adriamycin Human topoisomerase
Doxorubicin Human topoisomerase
6-mercaptopurine Human DNA polymerase
5-fluro uracil Thymidylate synthase
THANK YOU
Dr.Prashant Vishwanath
Biochemistry
JSS Medical College