[2] DNA AND GENOMICS

(a) Describe the structure and roles of DNA and RNA ie. mRNA,rRNA and tRNA

DNA and RNA are nuclei acids,considered as long-chained molecules or polymers that are universally found in all
organisms and are essential for life. Their subunits consist of repeating nucleotides. Each nucleotide consists of a
pentose sugar,an inorganic phosphate and a nitrogenous base.

DNA-Deoxyribose sugar
RNA-Ribose sugar

They differ in that one of the hydrogen atom at carbon 2 is replaced by a hydroxyl group in ribose.The phosphates give
the nucleotides their acidic property and is negatively charged.There are 4 different types of bases that can be present
in each nuclei acid. They are able to form hydrogen bonds with each other in a very specific manner.

Purines --> Adenine and Guanine

Pyrimidines --> Thymine, Cytosine and Uracil(ONLY found in RNA)

A total of 2 hydrogen bonds are formed between A and T(U) while a total of 3 hydrogen bonds are formed between C
and G.

Above is the basic structure of a nucleoside monophosphate.Note that the subunits used in DNA replication consists
of nucleoside triphosphates.

Formation of a nuclei acid:

2 nucleotides join to form a dinucleotide via condensation reaction.This occurs between the phosphate group of one
nucleotide and the -OH group of another nucleotide’s sugar group.A phosphodiester bond is formed between 3’ C and
5’ C

DNA structure:
Above is the structure of DNA.2 polynucleotide chains twist around each other to form a double helix.The 2 chains are
anti-parallel,one running from 5’ to 3’ and vice versa for the other.Each chain is made up of a sugar-phosphate
backbone and the bases project at right angles inwards.
Base-pairing is very specific,only between complementary bases.In one complete turn of the DNA helix(3.4 nm) there
will be 10 base pairs.The intertwined strands make 2 grooves of different widths-major and minor groove to facilitate
binding with specific proteins.

RNA structure:

Above-tRNA,rRNA and mRNA

RNA is a long polymer consisting of nucleotides joined by phosphodiester bonds.The bases include A,U,C and G.Sugar
is ribose instead of deoxyribose.Most RNA molecules are single stranded but this may not always be the case.They are
relatively small molecules and are chemically less stable and is thus,temporary.Note that the ratio of the bases may
also differ.

Roles of nuclei acids:

-Sequence of bases in gene/on DNA determine the amino acid sequence.
-Transcription takes place to form mRNA
-There are codons on mRNA
-Complementary base pairing A-U,C-G,T-A
-Carries the information for the protein to be synthesised
-Shine-Dalgarno sequence binds to small subunit of ribosome for proteins synthesis initiation
-Specific tRNA for each amino acid
-Has an anticodon for binding to mRNA codon
-3’ end has an attachment site for amino acid
-To bring the appropriate amino acid to the mRNA for protein synthesis
-ref CCA sequence on tRNA for binding of amino acid to it using amino-acyl tRNA synthase
-Complex of rRNAs and ribosomal proteins
-Form the large and small subunits of ribosomes
-Mention the role of A and P sites on ribosomes in translation

(b) Describe the process of DNA replication and the experimental evidence for semi-conservative replication

Semi-conservative replication:

Each strand of double stranded DNA serves as template for synthesis of daughter strand.Product consists of one
parental strand and one daughter strand.Important for the passing on of genetic information.

1) DNA replication begins at sites called origins of replication

2) Enzyme helicase breaks the hydrogen bonds b/w the complementary bases and the helicase than unwinds and
separates the 2 strands of DNA
3) Single-stranded DNA binding protein then attaches to the single-stranded DNA to stabilise them so that the
strands can be kept separated.Each strand acts as a template for synthesis of daughter strand.
4) As 2 strands separate,a replication bubble is formed with a replication fork at each end so DNA replication can
proceed in both directions.
5) Primase catalyses synthesis of a short RNA chain(RNA primer) that is complementary to one of the DNA
template.About 10 nucleotides long.
6) DNA polymerase(|||) then brings in free DNA nucleotides complementary to the exposed bases on the parental
strand and also the formation of the phosphodiester bond b/w 2 nucleotides.The new dsDNA grows as hydrogen
bonds form b/w complementary bases.
7) RNA primers are than excised and replaced with the correct DNA bases by DNA polymerase |

NB: DNA polymerase ||| cannot start DNA synthesis without any prior information of bases and it can only work in a
5’-3’ direction as it only adds DNA nucleotides the the free 3’-OH end.However,it is capable of proofreading and can
remove any incorrectly paired nucleotides.

8) Due to its limitations,one strand named the leading strand elongates continuously in the 5’-3’ direction ie DNA
polymerase moves towards replication fork.
9) Elongation of the other strand require the DNA polymerase to move along the template away from the replication
fork termed the lagging strand and replication is discontinuous.Short fragments-Okazaki fragments are formed
which are than joined together using DNA ligase. For the synthesis of each okazaki fragment,a new primer is
needed due to the absence of a free 3’-OH end.

Evidence:

The information on pg12 describes the growing of the bacteria so that data can be obtained for analysis. So
your account should include the experimental data obtained on pg 13.

Include the following:

 The density of the DNA extracted from G1 was compared with the density of DNA obtained from the G0
15
bacteria (made up entirely of N) as well as those DNA that contain nitrogenous bases that are made
14
up entirely of N.
15 15 14 14
It was found to form a band at a region that is in between that of the N N DNA and N N DNA.
15 14
This can be explained by the DNA being made up of a hybrid DNA ( N N) – one strand consisting of the
parental strand (made up of 15N) and the other consisting of the newly synthesized daughter strand
(made up of 14N). This hybrid DNA molecule will then have a density that is intermediate.
15 14
The DNA extracted from the G2 bacteria will then have a band in the intermediate density ( N N) as
14 14
well as a band in the N N. This is due to the both the 15N and 14N DNA of G1 being used as
templates. Since the nitrogenous bases that are available are of the 14N type, the newly synthesized
DNA should be of 2 types – one of intermediate type (with the 15N DNA strand as template) and the
other of the 14N type (using the 14N DNA strand as template).

(c)Describe how the information on DNA is used to synthesize functional polypeptide.

Reference: Lecture notes pg 5-6, (Mr Toh)

Protein synthesis involved 2 processes: Transcription and Translation.

2. Transcription is the process by which a sequence of bases in DNA template of a gene is copied by
complementary base pairing to form an mRNA.
(This occurs in the nucleus for the eukaryotic cell.)
Translation occurs where the information transcribed from DNA (=mRNA) directs the ordered polymerisation of
specific amino acids for the synthesis of polypeptides
(This occurs in the cytoplasm for the eukaryotic cell.)

Transcription
Initiation:
RNA polymerase attaches to the promoter region and the DNA double helix unwinds.
(Eukaryotic cell: 3 RNA polymerases)
Only the sense strand of the DNA will act as the template and be transcribed.
Elongation:
As RNA polymerase moves along the template from the 3’ to 5’ direction, free ribonucleoside
triphosphates (ATP, CTP, GTP, UTP) are being paired by complementary base pairing to the bases
on the sense strand.
(ii) i.e. if RNA polymerase reads an adenine, it will join a UTP to it.
(iii) The new RNA strand is synthesized in the 5' to 3' direction by the RNA polymerase. Adjacent
ribonucleotides are joined together by phosphodiester bonds.
Termination:
(i)Transcription stops after the reading of the termination sequence on the DNA and the RNA
polymerase dissociates from the DNA.
(ii) Newly formed mRNA is released and the DNA winds back.
(Eukaryotic cell: the first mRNA that is formed is known as the pre-mRNA and this will undergo processing:
capping at 5’ end, polyA tail added at 3’ end as well as RNA splicing before leaving the nucleus through
nuclear pores into the cytoplasm.)

Translation -Involves 4 main steps.

Preparation: Activation of amino acids and mRNA
The enzyme amino acyl-tRNA synthetase will catalyse the attachment of the amino acid to its respective
tRNA with its specific anticodon.

* Information on the mRNA are organised into groups of 3 bases (= 1 codon)

Initiation
Binding of small subunit to the mRNA via the Shine-Dalgarno sequence.
Movement of small subunit along mRNA until it locates the AUG initiation codon.
The tRNA with the formyl methionine (fMet-tRNA) binds to the initiation codon by complementary base
pairing.
The large subunit further binds, forming the initiation complex. (mRNA + fMet-tRNA + ribosome =
Initiation complex)
Each ribosome has 3 binding sites: A (Aminoacyl-tRNA binding) site, P (Peptidyl-tRNA binding) site and E
(exit site).
(Eukaryotic cell: the first aa-tRNA complex is met-tRNA i.e. methionine is not methylated.)

Elongation
Amino acids are added on, one at a time, to a growing polypeptide chain; order specified by base
sequence on mRNA
After the initiation complex is formed, the codon adjacent to the initiation codon now occupies the
A site.
A second aa-tRNA molecule (with the complementary anticodon) then attaches itself to this codon
in the A site.
Peptidyltransferase or peptide synthetase catalyses the formation of a peptide bond formed
between amino acid occupying the P site and amino acid occupying A site.
Translocation occurs where there is a relative movement of the ribosome and the mRNA (5’ to 3’
direction). This results in
1. The tRNA in the P site (without its amino acid) moving to the E site;
2. The dipeptidyl-tRNA in A site moves to the Psite and
3. The next codon is now positioned on the A site.
The process repeats and the polypeptide chain elongates

d) Termination
(i) Elongation stops when the ribosome reaches a ‘stop’ codon (UAA, UAG, UGA).
(ii) The ribosome accepts a release factor. The release factor causes the addition of water molecule that
results in the release of the polypeptide.
(iii)The ribosomal subunits and other components of the assembly dissociates.
(iv)The mRNA being labile, breaks down.

(Prokaryotic cell: Transcription coupled to translation.)

(d)Describe how a change in the sequence of the DNA nucleotide (gene mutation) may affect the amino acid sequence
in a protein and hence the phenotype of the organism eg. sickle cell anaemia.

Gene mutation can be defined as an inherent,chemical change in the base sequences in one gene and may involve a
change in one or more bases.If only one base is involved,it is a point mutation.

Deletion: One or more nucleotides are removed from a sequence of bases.

Insertion: One or more nucleotides are added to a sequence of nucleotides.

Substitution: When a nucleotide gets replaced by another.

Inversion: When a sequence of nucleotides becomes separated by the allele and rejoins at the original position but is
inverted.

The first 2 are examples of what we termed as frameshift mutations.This is because it causes a change in the reading
frame of the sequence of bases. Often results in non-functional protein as ribosomes begin to read incorrect codons
from point of insertion or deletion.IF it is deletion/insertion of 3 nucleotides,a codon that is,the effect is not as drastic
as it merely involves one amino acid being added/removed from original polypeptide.
HOWEVER,please note that this is only a viable argument if it is inserted/deleted as a codon ie together because if it is
insertion/deletion of 3 bases randomly and separately in the gene than the argument may not be valid.

Usually the effect of substitution is not as serious as replacing a nucleotide may not necessarily alter the amino acid
residue of a polypeptide. There is little effect if the substitution doesn’t take place at critical regions,such as the
sequence coding for the active site of an enzyme.There is drastic effect if for instance,it causes the conversion of a
codon that is originally coding for something into a stop codon.

Reference: Lecture notes pg 22 (Mr Toh)

Must know the actual change in the base, the amino acid as well as the effect of the change on the property
of haemoglobin such as decreased solubility and precipitation as fibers resulting in the RBC being sickle
shape.