7.

1 DNA STRUCTURE AND REPLICATION

DNA structure suggested a mechanism for DNA replication

Franklins x-ray diffraction experiments demonstrated that the DNA helix is
both tightly packed and regular in structure
Watson and Crick found that the tight packing was occur if a pyrimidine
was paired with a purine and if the bases were upside down
Adenine has a surplus negative charge and thymine has a surplus positive
charge so the pairing was electrically compatible
o Pairing cytosine with guanine allows for the formation of 3 hydrogen
bonds which enhances stability
The nitrogenous bases were relatively hydrophobic in comparison to the
sugar-phosphate backbone so were likely positioned in the interior of the
helix
DNA structure suggests two mechanisms for DNA replication:
o Replication occurs via complementary base pairing (adenine pairs
with thymine, guanine pairs with cytosine)
o Replication is bi-directional (proceeds in opposite directions on the
two strands) due to the antiparallel nature of the strands

Nucleosomes help to supercoil DNA

A nucleosome consists of a central core of eight histone proteins
(octamer) with DNA coiled around the protein
The octamer consists of two copies of 4 different types of histones
o Histones have a tertiary structure
The association of histones with the DNA allows for supercoiling
Supercoiling allows a great length of DNA to be packed into a smaller
space inside the nucleus
Nucleosomes are linked by an additional histone protein (H1 histone) to
form a string of chromatosomes
o Linker DNA helps stick DNA to the histones
These then coil to form a solenoid structure (6 chromatosomes per turn)
which is condensed to form a 30 nm fiber
These fibers then form loops, which are compressed and folded around a
protein scaffold to form chromatin
Chromatin will then supercoil during cell division to
form chromosomes that are visible (when stained) under microscope
If DNA is supercoiled it cannot be transcribed. Keeps it from making
proteins constantly all the time

DNA replication is carried out by a complex system of enzymes

 Single stranded binding proteins prevents single stranded DNA that has
been opened by helicase and prevents it from reforming a double helix
Steps of DNA replication with protein functions:
o DNA Helicase unwinds and unzips the base pairs by breaking the
hydrogen bonds
o DNA polymerase III makes a complementary strand on the leading
strand by adding deoxynucleoside triphosphate to the 3 end of the
complementary strand
o RNA primase follows helicase, leaving RNA primers that are markers
for initiation of DNA Polymerase III
o DNA polymerase attaches to an RNA primer and replicates DNA
o When it reaches another RNA primer, it detaches and leaps to the
next primer
o DNA polymerase I moves along the replication form removing and
replacing the RNA primers
o DNA ligase attaches the Okazaki fragments into a continuous strand
of DNA

DNA polymerase can only add nucleotides to the 3 end of a

primer
o DNA polymerase cannot initiate replication, it can only add new
nucleotides to an existing strand
o For DNA replication to occur, an RNA primer must first be
synthesized to provide an attachment point for DNA polymerase
o The phosphate group of new DNA nucleotides is added to the 3
carbon of the deoxyribose at the end of the chain extending the
chain in a 5 to 3 direction

DNA replication is continuous on the leading strand and

discontinuous on the lagging strand
One strand of DNA polymerases can move in the same direction the
replication fork so replication is continuous. This is the leading strand
In the lagging strand, the DNA polymerases have to move opposite the
replication fork, so replication is discontinuous.
o lagging strand is copied as a series of short fragments (Okazaki
fragments), each preceded by a primer
o The primers are replaced with DNA bases and the fragments joined
together by a combination of DNA pol I and DNA ligase
 Some regions of DNA do not code for proteins but have other
important functions
Coding sequences: the parts of DNA that codes for polypeptides
Some non-coding sequences have functions:
o A guide to produce tRNA and rRNA
Promoters are binding sites for RNA polymerase
Operators are binding sites for repressor proteins that
prevents transcription
o Play a role in the regulation of gene expression such as enhancers
and silencers
Enhancers increase transcriptions
Silencers inhibit transcription
Repetitive sequences can be common in eukaryotes
2 types of repetitive sequences
o Moderately repetitive sequences
o Highly repetitive sequences (satellite DNA)
Repetitive sequences occurs on the ends of chromosomes called
telomeres which serves a protective function

Rosalind Franklins and Maurice Wilkins investigation of DNA

structure by X-ray diffraction
Diffraction: scattering of X ray beams when directed at a material.
DNA was arranged to make most of the strands parallel so that diffraction
occurs in a regular way.
An X-ray detector is placed close to the sample to collect the scattered
rays.
Diffraction patterns is recorded using X-ray film.
From the scattering pattern produced by a DNA molecule, certain
inferences could be made about its structure
o Composition: The cross in the center of the pattern indicated that
DNA is a double stranded molecule. The angle of the cross shape
showed the steepness of angle of the helix
o Orientation: Nitrogenous bases are closely packed together on the
inside and phosphates form an outer backbone
o Shape: The DNA molecule twists at regular intervals (every 34
Angstrom) to form a helix (two strands = double helix)

Use of nucleotides containing dideoxyribonucleic acid to stop DNA

replication in preparation of samples for base sequencing
 DNA sequencing refers to the process by which the base order of a
nucleotide sequence is clarified
Dideoxynucleotides (ddNTPs) lack the 3-hydroxyl group necessary for
forming a phosphodiester bond so it prevents further elongation of a
nucleotide chain and effectively terminate replication
The resulting length of a DNA sequence will reflect the specific nucleotide
position at which the ddNTP was incorporated
Dideoxynucleotides can be used to determine DNA sequence using the
Sanger method
o Four PCR mixes are set up, each containing stocks of normal
nucleotides plus one dideoxynucleotide (ddA, ddT, ddC or ddG)
o As a typical PCR will generate over 1 billion DNA molecules, each
PCR mix should generate all the possible terminating fragments for
that particular base
o When the fragments are separated using gel electrophoresis, the
base sequence can be determined by ordering fragments according
to length
o If a distinct radioactive or fluorescently labelled primer is included in
each mix, the fragments can be detected by automated sequencing
machines
o If the Sanger method is conducted on the coding strand (non-
template strand), the resulting sequence elucidated will be identical
to the template strand

Tandem repeats are used in DNA profiling

DNA profiling is a technique by which individuals can be identified and
compared via their respective DNA profiles
Within the non-coding regions of an individuals genome there exists
satellite DNA long stretches of DNA made up of repeating elements
called short tandem repeats (STRs)
Tandem repeats can be extract by using restriction enzymes and then
separated with gel electrophoresis for comparison
As individuals will likely have different numbers of repeats at a given
satellite DNA locus, they will generate unique DNA profiles
Longer repeats will generate larger fragments, while shorter repeats will
generate smaller fragments

Analysis of results of the Hershey and Chase experiment

providing evidence that DNA is the genetic material
In the mid-twentieth century, scientists were still unsure as to whether
DNA or protein was the genetic material of the cell
Vector: something that carries something else from point A to point B
o Mosquitos are vectors of Dengue
o Using the virus as a vector
Viruses were grown in an isotopic medium to radioactively label a specific
viral component
o Viruses grown in radioactive sulfur (35S) had radiolabelled proteins
(sulfur is present in proteins but not DNA)
Radioactive so that they can trace / identify it
 o Viruses grown in radioactive phosphorus (32P) had radiolabeled DNA
(phosphorus is present in DNA but not proteins)
The viruses were then allowed to infect a bacterium (E. coli)
the virus and bacteria were separated via centrifugation
The larger bacteria formed a solid pellet while the smaller viruses
remained in the supernatant (the clear liquid floating on the surface)
The bacterial pellet was found to be radioactive when infected by the 32P
viruses (DNA) but not the 35Sviruses (protein)
This demonstrated that DNA, not protein, was the genetic material
because DNA was transferred to the bacteria

7.2 TRANSCRIPTION AND GENE EXPRESSION

Understandings:

Transcription occurs in a 5 to 3 direction

RNA polymerase covalently binds the NTPs together in a reaction that
involves the release of the two additional phosphates
The 5-phosphate is linked to the 3-end of the growing mRNA strand,
hence transcription occurs in a 5 3 direction
The process of transcription can be divided into three main steps:
initiation, elongation and termination
o In initiation, RNA polymerase binds to the promoter and causes the
unwinding and separating of the DNA strands
o Elongation occurs as the RNA polymerase moves along the coding
sequence, synthesizing RNA in a 5 3 direction
o When RNA polymerase reaches the terminator, both the enzyme
and nascent RNA strand detach and the DNA rewinds
Nucleosomes help to regulate transcription in eukaryotes
Eukaryotic DNA is wrapped around histone proteins to form compact
nucleosomes
These histone proteins have protruding tails that determine how tightly
the DNA is packaged
 Acetylation: Adding an acetyl group to the tail neutralizes the charge,
making DNA less tightly coiled and increasing transcription
Methylation: Adding a methyl group to the tail maintains the positive
charge, making DNA more coiled and reducing transcription

Condensed heterochromatin: When DNA is supercoiled and not accessible

for transcription
o Methylation inhibits transcription
Euchromatin: When the DNA is loosely packed and therefore accessible to
the transcription machinery
o Acetylation promotes transcription
Eukaryotic cells modify mRNA after transcription
The immediate product of mRNA transcription is referred to as pre-mRNA
It must go through several stages of post-transcriptional modification to
become mature mRNA
3 post-transcriptional events
o Capping: involves the addition of a methyl group to the 5-end of
the transcribed RNA
o Polyadenylation describes the addition of a long chain of adenine
nucleotides (a poly-A tail) to the 3-end of the transcript
o Splicing: removing introns (mRNA sequences that do not code for
proteins)
The remaining coding portions of the mRNA are called exons.
These will be spliced together to form the mature mRNA
Splicing of mRNA increases the number of different proteins an
organism can produce
Splicing can also result in the removal of exons a process known as
alternative splicing
The selective removal of specific exons will result in the formation of
different polypeptides from a single gene sequence
 Gene expression is regulated by proteins that bind to specific
base sequences in DNA
Necessary proteins are expressed in an unregulated fashion
The expression of other proteins that are produced only at certain times
needs to be regulated
Enhancers: regulatory sequences on the DNA which increase the rate of
transcription when proteins bind to them
Silencers: the sequences on the DNA which decrease the rate of
transcription when proteins bind to them
Promoter-proximal elements: binding of proteins to them is necessary to
initiate transcription
The environment of a cell and of an organism has an impact on
gene expression
Changes in the external or internal environment can result in changes to
gene expression patterns
o Chemical signals (morphogens) within the cell can trigger the
expression of gene
o This allows gene expression to change in response to alterations in
intracellular and extracellular conditions
examples of organisms changing their gene expression patterns in
response to environmental changes
o Hydrangeas change color depending on the pH of the soil
o Humans produce different amounts of melanin (skin pigment)
depending on light exposure
o Certain species of fish, reptile and amphibian can even change
gender in response to social cues

7.3 TRANSLATION
The use of molecular visualisation software to analyse the
structure of eukaryotic ribosomes and a tRNA molecule
Structure of ribosome includes
o Proteins and rRNA
o One large sub-unit, one small
o 3 binding sides for tRNA but only 2 can bind at the same time
E site
 P site
A site
o Binding site for mRNA on the surface
Initiation of translation involves assembly of the components that
carry out the process
mRNA molecule binds to the small subunit of the ribosome at an mRNA
binding site (P site)
a tRNA molecule carrying methionine binds o the start codon AUG
The large subunit then binds to the small subunit
Another tRNA binds at the A site and a peptide bond is formed between
the amino acids in the P and A site
Synthesis of the polypeptide involves a repeated cycle of events
Elongation occurs through a series of repeated steps
The tRNA in the P site moves to the E site, freeing it
This allows another tRNA to occupy the vacant A site
Disassembly of the components follows termination of translation
Elongation continues until a stop codon is reached and the polypeptide is
released
Free ribosomes synthesize proteins for use primarily within the
cell
Proteins are either synthesized in the cytoplasm or at the endoplasmic
reticulum depending on the final destination of the protein
Translation occurs more commonly in the cytosol
Proteins used in the cytoplasm, mitochondria, and chloroplasts are
synthesized by free ribosomes in the cytoplasm
Bound ribosomes synthesize proteins primarily for secretion or for
use in lysosomes
Proteins are will be used in the ER, Golgi apparatus, lysosomes, plasma
membrane or outside the cell are synthesized by ribosomes bound to the
ER
Translation can occur immediately after transcription in
prokaryotes due to the absence of a nuclear membrane
In eukaryotes, there is a delay between transcription and translation due
to the mRNA having to exit the nucleus
In prokaryotes, as soon as the mRNA is transcribed, translation begins
The sequence and number of amino acids in a polypeptide is the
primary structure
The secondary structure is the formation of alpha helices and
beta pleated sheets stabilised by hydrogen bonding
The tertiary structure is the further folding of the polypeptide
stabilised by interactions between R groups
The quaternary structure exists in proteins with more than one
polypeptide chain