Replication- replication is the process of producing two identical copies of DNA from one parent

strand. Dna replication is a semi conservative produce, where each newly synthesised DNA stand
contains one parent strand and one daughter strand. DNA molecule is a double helix structure and
contains two complementary strands

Requirements for DNA replication

- Nucleotides
- DNA template
- RNA primer
- Enzymes
- Proteins

Initiation

- DNA replication starts at the origin of replication (OriC)

- DNA A protein recognises OriC and will bind to that site, denaturing DNA
- Helicase will then bind to oric and start rewinding the complementary dna strands
- This results in the formation of a replication fork
- SSB proteins will stabilise the separated strands and prevent the single strabnds from
rewinding
- DNA primase will add primer, which is a short RNA segment, to the 3’ end of dna template
- Primer is the starting point for addition of dna nucleotides

Elongation

- DNA strands are antiparallel, where one runs from 3-5 and other runs from 5-3
- DNA polymerase recognises RNA primer at 3 end and will start adding nucleotides at 3 end
- DNA synthesis is always from 5-3 direction as DNA polymerase only adds nucleotides at 3
end
- This resukts in the production of two different strands: leading and lagging strands
- The leading strand uses 3-5 parent strand as template, synthesis occurs continuously in the
5-3 direction towards the replication fork. Uses ony one rna primer
- Lagging strand uses 5-3 parent strand as template and synthesis occurs discontinuously from
the 5-3 direction away from the replication fork. Several rna primers are present. Okazaki
fragments formed
- When synthesis of both strands are completed, exonucleas will remove rna primer, and DNA
polymerase will replcase it with correct nucleotides
- Dna ligase will also join the okazaki fragments of the lagging strands together and form one
continuous strand of DNA

Termination

- Termination occurs when replication forks from 2 active replication origins collide

Proofreading

- Incorrect/mismatched bases are removed by nuclease

- DNA polymerase adds correct nucleotides, and dna ligase will join the nucleotides together
Transcription- transcription is the process where an rna strand is transcribe from a antisense DNA
strand as template. It’s the first step leading to gene expression

DNA contains two types of strands:

- Sense strand which is the coding strand. It runs from 5 to 3 direction

- Antisense strand is the template strand which runs from 3 to 5 direction

Process of transcription

Initiation

- Promoter signals the initiation of rna synthesis

- Transcription factors help RNA polymerase to recognise promoter sequence- TATA box, and
will bind to it
- Once recognised the TATA box, RNA polymerase will start unwinding the DNA double helix
and provide the single strand DNA template needed for transcription

Elongation

- RNA polymerase moves along the antisense dna template strand and synthesises a single
strand of RNA by adding nucleotides from the 5-3 direction
- DNA unwinds ahead of RNA polymerase, and will rewind as double helix behind rna
polymerase
- Rna polymerase unwinds 10-20 nucleotides at a time
- Rna polymerase adds nucleotides in the 5-3 direction

Termination

- Transcription stops when rna polymerase recognises terminator sequence AAUAAA

- Termination of transcription is by endonucleolytic cleavage
- Rna strand is released and rna polymerase dissociates from dna
- This will produce immature rna which goes through more processing in post transcriptional
modification

Post transcriptional modification

- To allow rna molecule to be recognised by molecules that mediate rna translation into
protein
- Portions of rna chain that are not neccesory are removed

Involves: adding of protective bases at ends of RNA and splicing

- 5’ capping- addition of modified guanine to the 5 end of rna

- To promote rna for translation; 5’ is required for binding of ribosomes and translation
initiation factor
- To prevent mrna from degradation by exonucleases. This increases half life of mrna as
transporting mrna requires time
- 3 poly A tailing- addition of 50-250 adenine at 3 end of mrna
- To promote export of mrna from nucleus
- To protect mrna from enzymatic degration in the cytoplasm
- Splicing is the removal of introns from mrna. Introns are non coding sequences an exons are
coding sequences. Introns are removed and exons are joined back together
Reverse transcription is the synthesis of DNA from RNA by RNA dependent DNA polymerase- reverse
transcriptase. Used by viruses to replicate their genomes

Translation- transmission of genetic information from mrna into protein

Genetic code- the relationship between sequence of nucleotides in mrna transcrips and sequence of
amino acid in protein. Genetic sequence is encoded as a sequence of codons. Each amino acid is
specified by a codon

Phenylketonuria- failure to process phynylalanine to make tyroxine due to mutation in enzyme

phenylalanine hydroxylase. Accumulation of ohynylalanine results in brain intellectual disability

Alkaptonuria- defects in enzyme homogentisic acid oxidase, which is an enzyme in tyrosine

degrasation pathway. Results in accumulation of homogentisic aciduria. Causes large joint arthritis,
dense, black pigment deposits on intravertebral disc of vertebrae

Albinism- defect in tyrosine metabolism resulting in deficiency of melanin production. Causes

hypopigmentation of skin, hair, eye

Argininosuccinate acidurea- dysfunctional argininosuccinate layse, which is an enzyme needed to

convert argininosuccinate into arginine. Arginine converts to ornithine to initiate urea cycle. If
arginine not synthesised, urea cycle not initiated, nitrogen is built up and not expelled. This causes
ammonia accumulation which is toxic

Amino acidurea- the urinary excretion of amino acid due to defect in amino acid metabolism. Leads
to mental retardation

Importance of mitochondrial structure in ATP production: energy released by oxidation of nutrients

is used in the form of chemical energy ATP. Production of ATP in mitochondira is the result of
oxidative phosphorylation

Complex 1- nadh coq oxidoreductase

- Catalyses transfer of electron from nadh to coq. This complex has Flavin mononucleotide

Complex 2- succinate coq oxidoreductase

- Catalyses the oxidation of succinate to fumarate in the TCA cycle to produce FADH2. FADH2
tarnsfers electron to coq

Complex 3- cytochrome c reductase

- Catalyses oxidation of reduced coq, electrons are passed along to cyc in multiple process

Complex 4- cytochrome c oxidase

- Catalyses final step of electron transport, transfer electron form cytofchrome C to oxygen,
which is the final electron acceptor
- Proton pumping occurs
- Cytochrome c oxidase is an integral part of inner mitochorndiral membrane
Replication- process which produces two identical copies of DNA from one parent dna strand

Replication is a semiconservative process in which each newly synthesisied strand contains one
parent strand and one daughter strand

Why is replication important? Replication is an essential process because whenever a cell divides,
two new daughter cells must contain the same genetic information as the parent cell.

Initiation

- Replication starts at OriC- origin of replication

- DNA A protein recognises oric and will bind to it, denaturing the dna
- Helicase will bind to oric and start unwinding the double helix dna strand
- Ssb proteins will help stabilise the separated strands and prevent them from rewinding
- Dna primase adds primer to the 3 end of dna,
- Primer is a short segment of rna molecule which indicates the starting point of dna
nucleotide addition

Elongation

- dna contains double strands which are antiparallel, where one runs from 5 to 3 and the
other runs from 3 to 5
- dna polymerase will add nucleotide at the 3 end where primer is located
- therefore dna synthesis is always from 5 to 3
- this leads to the formation of two strands
- leading strand- uses 3-5 parent strand as template, dna synthesis runs from 5-3 direction
continuously towards the replication fork. It only uses one rna primer
- lagging strand- uses 5-3 dna parent strand as template, dna synthesis runs from 5-3 direction
discontinuously away from replication fork. Uses several rna primers. Okazaki fragments are
produced
- once both strands are produced, exonuclease will remove rna rpimer from the strand.
- dna polymerase will replace primers by adding in the correct nucleotides
- dna ligase will join the okazaki fragments together to form a continuous strand

termination

- dna replication stops when two replication forks from two active origins collide

proofreading

- there may be addition of mismatched/incorrect nucleotides

- nuclease enzyme will remove the wrong nucleotides
- dna polymerase will add correct ones
- and dna ligase will join them back together
Transcription- process of transcribing genetic information from dna template strand into mrna. It’s
the first step to gene expression. Transcription uses antisense dna strand as template

Dna is double stranded, which conatins sense strand and antisense strand

- sense strand runs from 5-3 direction, which has the same nucleotide seuqnece as rna
- antisense strand runs from 3-5 direction, and is used as a template in transcription

difference between replication and transcription

Replication Transcription
Uses dNTP as substrate Uses NTP as substrate
Has primer No primer
DNA polymerase enzyme RNA polymerase
Product is dna Produce is rnna
Base pair= ATCG AUCG

Initiation

- transcription is initiated by promoter

- transcription factors help rna polymerase recognise promoter sequence, TATA box and will
bind to it
- rna polymerase binds to promoter region and will start unwinding the double helix DNA
- this produces single stranded dna needed for template in elongation

elongation

- rna polymerase moves along the antisense strand of dna

- rna polymerase will add dna nucleotides to the 3 end of dna template, therefore rna
synthesis is from 5 to 3
- dna strands are unwinding ahead of rna polymerase, and is rewinding behind it
- rna polymerase unwinds 10-20 dna bases at a time
- rna polymerase adds nucleotide in the 5 to 3 direction

termination

- transcription stops when rna polymerase reaches terminator sequence

- terminator sequence is AAUAAA
- termination is performed by endonuclelytic cleavage, which releases the newly synthesised
rna strand and dissociate rna polymerase from antisense dna strand
- the rna formed is known as pre-mrna which requires further modifications

post transcriptional modification

- mrna strand needs to be further modified to allow recognition by translational molecules

and to remove unwanted parts of mrna
- involves addition of protective bases at the ends of mrna and splicing
- 5’ capping- addition of modified guanine at 5 end of mrna
- This helps promote translation by allowing ribosome to bind at 5 end of the mrna
- This also helps prevents degradation by exonuclease, which increases the half life of mrna as
mrna needs time to be exported from the nucleus
 - 3 poly A tailing- addition of 50-250 adenine at 3 end of mrna. This helps promote mrna
exportation from the nucleus into the cytoplasm and prevents enzymatic degradation of
mrna in cytoplasm
- Splicing- removal of introns. Mrna contains introns, non coding sequences, and exons,
coding sequences. Introns need to be removed, and exons are joined back together

Reverse transcription- synthesis of dna from rna template driven by reverse transcriptase enzyme

- Used by viruses to replicate their genome

Translation- the transmission of genetic information from mrna to protein. It’s the second stage in
gene expression. Translation occurs in the cytoplasm where ribosomes are located

Mrna- messenger rna which caries copies of instructions for assembling of amino acid into protein

Rrna rich ribosomes- made of rrna and protein. Contains 2 subunits. Small subunit binds to mrna first
then followed by large subunit. Contains 3 major sites: A site which accommodates incoming
aminoacyl trna; P site which accommodates peptidyl trna and E site which accommodates
deacylated trna which is about to leave the ribosome

Trna- carries amino aicd to ribosome

Initiation

- Small ribosomal subunit binds to 3 end of mrna and will move along it until it reaches a start
codon AUG
- Initiator aminoacyl trna molecule carrying methionine will bind to the start codon with its
anticodon UAC
- Large ribosomal subunit will then assemble itself to trna at P site
- It forms complete ribosomal complex with small ribosomal subunit

Elongation

- A second aminoacyl trna will bind to the next codon at A site

- Amino acid at A site and at P site will covalently attach via a peptide bond. This is the
transpeptidation process
- Trna at P site is now deacylated, while trna at A site will carry the peptide chain. Trna at A
site forms peptidyl trna
- Deacylated trna will move into E site and is released, and peptidyl trna will move into P site.
This is translocation process
- A site is now unoccupied and ready to receive the next trna molecule

Termination

- Elongation and translocation is terminated when ribosome reaches a stop codon (uaa, uag,
uga)
- Stop codons are recognised by release factors, which binds to the stop codon at A site
- This leads to the release of newly synthesised polypeptide chain, and also the dissociation of
ribosome into two separate subunits
Post translational modification

- Proteolytic cleavage/trimming
- Covalent modification
- Ubiquination

Proteolytic cleavage

- removal of a part of the translated sequence

- example: conversion of proinsulin to insulin
- to convert the 84 residue proinsulin into insulin, the internal residue c chain must be
removed
- it has to be cleaved into 2 polypeptide chains and about 30 amino acids must be removed to
form insulin

covalent modification

- it’s the addition of new functional groups to the protein

- for example- glycosylated haemoglobin (HbA1C)
- glycosylated haemoglobin is the haemoglobin which is bound to glucose
- hba1c test will measure the percentage of hbaic in blood, which can be used to diagnose
diabetes or monitor diabetes

ubiquination

- ubiquitin is a small regulatory protein which is attached to protein and will label them for
destruction
- protein that are defective are often marked for degradation
- giant proteosomes will recognise the tagged proteins and destroy them

genetic code

- the relationship between the sequence of nucleotides in mrna transcript and sequence of
amino acid in proteins
- genetic information is encoded as a sequence of codons
- each codon codes for an amino acid