Replication – Transcription – Translation DNA REPLICATION

DNA → mRNA → Protein Replication –

3 Challenges:

Central Dogma • Two DNA strands are separated from

each other
• Replication – duplication of DNA, giving rise
to new DNA molecule – The cell must protect the unwound
portions from nucleases – as they
• Transcription – process of making RNA
may attack the single stranded DNA
from DNA
– Synthesizing of DNA from 5’ to 3’
• Translation – RNA sequence of mRNA is
end
used to direct the synthesis of proteins
– Newly formed DNA has 2
antiparallel strands:

– Template strand is 5’ to 3’

– New strand is 3’ to 5’

– Guarding against error of

replication

Semiconservative Replication
• Nearly all organism follow the genetic flow • DNA replication involves separation of 2
DNA → RNA → Protein original strands and production of 2 new
strands with the original strands as
• The only major exceptions are some viruses
templates.
• Retrovirus
KEY ENZYMES
– RNA is the genetic material instead
of DNA
Helicase helix-
– RNA can direct its own synthesis destabilizing
protein
– Reverse transcriptase catalyzes
this process
DNA Gyrase prevents
(Class II positive
topoisomerase) supercoiling
HIV

➢ Infection by Retroviruses Primase catalyzes the

synthesis of
➢ Reverse transcriptase is a target for drug RNA primer
design
 DNA replicates DNA – Single-stranded DNA are
Polymerase molecules to susceptible to degradation by
form the new nucleases
strand – Single-Strand Binding (SSB)
Protein binds tightly to the single-
DNA Ligase glues that stranded portions, protecting them
fragments from degradation
together

Step 2: PRIMING

• Primase catalyzes the synthesis of an RNA

primer
DNA Replication
– copies a short stretch of the DNA
• DNA double helix unwinds at a specific
template producing the RNA primer
point – origin of replication
sequence
• DNA synthesis is bidirectional.
– Primer has a free 3’hydroxyl to which
– exception are some viruses and the growing chain can attach
plasmids
Step 3: FORMATION OF THE NEW STRAND
– Two replication forks moves in
• DNA Polymerase III starts the synthesis of
opposite directions -- antiparallel
2 new strands
– Replication forks – two points of
• Newly formed DNA is linked to the
each origin of replication
3’hydroxyl of the RNA primer

• DNA Polymerase I – repair function

– removes the RNA primer

(exonuclease activity) and

– replaces it with deoxynucleotides

(polymerase activity)

DNA POLYMERASES

DNA - First polymerase

Polymerase discovered
Step 1: UNWINDING OF THE DOUBLE HELIX
I
- Consists of a single
• Helicase unwinds the double-helix
polypeptide chain
– Unwinding of the helix introduces
Function: Synthesis,
positive supercoiling
Proofreading, Repair
– Positive supercoiling will make
further replication impossible DNA -Not required for replication
Polymerase
– DNA Gyrase places negative Function: Repair
II
supercoils ahead of the replication
forks
 DNA - Consists of a core enzyme • The 3’-hydroxyl group at the end of the
Polymerase responsible for the growing DNA chain is a nucleophile
III polymerization and 3’ • It attacks at the phosphorus adjacent to the
exonuclease activity sugar in the nucleotide to be added to the
Function: Synthesis of new growing chain
strands – eliminating the pyrophosphate and

DNA Function: Unique repair a new phosphodiester bond is formed

Polymerase mechanism – SOS Response
IV

DNA
Polymerase
V

Human Immunodeficiency Virus

PROOFREADING AND REPAIR ➢ Infection by retroviruses

• Accuracy of the replication process should ➢ Reverse transcriptase is a target for drug
be high to prevent mutations. design

• Proofreading – refers to the removal of ➢ Anti-retroviral Drugs (ARV) such as AZT and
incorrect nucleotides immediately DDI lacks hydroxyl group at the 3’-position

– Improves the fidelity of replication ➢ They cannot form phosphodiester linkages

to one error in every 109 to 1010 base found in the nucleic acids
pairs ➢ Thus, interfering in the replication of the
• Replication resumes when the correct virus by preventing nucleic acid synthesis
nucleotide is added

Step 4: FINAL LINKING OF THE NEW STRAND

• DNA Ligase is responsible for linking the

small fragments of DNA

ADDITION OF NUCLEOTIDE TO A GROWING

CHAIN:
 • Base-excision repair

– A base that has been damaged by

oxidation or chemical modification
is removed by DNA glycosylase

• Nucleotide-excision repair

– Common for DNA lesions caused by

ultraviolet or chemical means –
After DNA Replication leading to deformed DNA
structures
What if a mutation managed to escape?
NUCLEOTIDE EXCISION REPAIR
• Mismatch-Repair System

– Repair system identifies which of the

two strand is the correct one

– Area with mismatch is removed

– DNA polymerases replicate the area

again

BASE EXCISION REPAIR

TRANSCRIPTION AND TRANSLATION

Transcription

• Process of using a DNA template to

produce RNA

• Major control point in the expression of

genes and the production of proteins

• Produces all types of RNA

 – mRNA, tRNA, rRNA, snRNA, miRNA, • RNA polymerase initiates RNA synthesis
and siRNA

DNA Strands

• Template strand

– Directs the synthesis of RNA

– Its code complements the RNA

produced

– Coding strand

– Its sequence of DNA will be the
same as the RNA sequence
produced
• With the exception of U
replacing T
– RNA sequence is used to determine
what amino acid is produced ( in the
case of mRNA)
starting at the starting point and moves
along the gene
– Reads the template strand from 3’ to
5’
– Generates RNA from 5’ to 3’
– RNA nucleotide is attached to the 3’
– RNA polymerase zips the DNA back
up as it goes

– Only 10 to 20 bases are exposed at

a time

Step 1: Chain Initiation

• RNA polymerase binds to the promoter

forming closed complex
Step 3: Chain Termination
• Does not need a primer
• At this point, RNA polymerase reached the
end of the gene

– RNA polymerase detaches and

– DNA is returned to its original state

– mRNA is produced at this time –

which carries the information coded
in the gene
Step 2: Chain Elongation
 – mRNA then leaves the nucleus and
moves to the ribosome where
translation occurs
TRANSLATION OF THE GENETIC MESSAGE
Protein Biosynthesis
• Protein biosynthesis requires ribosomes,
mRNA, tRNA, and protein factors
– Ribosome – site of protein synthesis
– mRNA & tRNA – responsible for the
correct order of amino acids in the
growing protein chain
– mRNA acts as a code for a specific
protein
Genetic Code
• Most important feature of the code: triplet,
nonoverlapping, commaless, degenerate,
universal code
• Codon
– ‘Triplet code’ that specifies one
amino acid
– 64 possible codons and each
uniquely encodes the 20 amino
acids
– Forms base pairs with
complementary anticodon
– Anticodon
– carried by a specific tRNA
– Each tRNA is covalently linked to a
particular amino acid
Amino Acid Activation
• Before an amino acid is incorporated into a
growing chain, it must first be activated
• aminoacyl-tRNA synthetases activates the
amino acid in two steps:
– Amino acid is covalently bond to
adenine nucleotide producing
aminoacyl-AMP
Then binds to tRNA forming aminoacyl-tRNA
TRANSLATION
Step 1: Chain Initiation
• Translation occurs inside the ribosome
 • Small ribosomal subunit binds to mRNA – Forming a dipeptidyl-tRNA at the A
and an initiator tRNA – which adheres to the site and an uncharged tRNA (tRNA
start codon with no amino acid) at the P site

• Large ribosomal subunit to complete the – Translocation

translation initiation complex
– Uncharged tRNA moves from P site
to E site where it is released

– Peptidyl-tRNA moves from the A site

to the vacated P site

– As tRNAs enters and exits the

ribosome, a polypeptide chain is
formed

– This process is repeated until the

Step 2: Chain Elongation
stop codon is reached
• There are 3 tRNA binding sites:

– P site (peptidyl)

• binds to tRNA that carries a

peptide chain

– A site (aminoacyl)

• binds incoming aminoacyl-

tRNA

– E site (exit)

• carries an uncharged tRNA

that is about to be released
from the ribosoome

Step 2: Chain Elongation Step 3: Chain Termination

• P site is the first to be occupied • Stop codon is required for the termination
of protein synthesis
– Starting with the start codon which
contains the amino acid methionine – UAA, UAG, UGA

3 steps: – The completed polypeptide

detaches and leaves
• Aminoacyl-tRNA binding
– Proceeds to protein folding and
– The second aminoacyl-tRNA binds modification
to the A site

– The amino acid it carries covalently

binds to the methionine

– Peptide bond formation

– α-amino group of the amino acid

forms a nucleoohilic attack on the
carbonyl group of the in the P site