TUTORIAL: DNA BIOLOGY AND TECHNOLOGY

1. Describe the biochemical composition, structure and replication of DNA.

DNA is a polymer where the monomer units of DNA are nucleotides and the
polymer is known as polynucleotide. DNA molecule consists of two strands of
polynucleotide that wound together into a double helix. In the two polynucleotide strands
is a phosphate group linked together by a phosphodiester bond between the phosphate
group of one nucleotide and the hydroxyl group of carbon 3 of the next nucleotide that is
3’ terminal while at one end of each strand is a phosphate group linked to the carbon 5 of
deoxyribose sugar. This is the 5’ terminal of each strand. The 3’ and 5’ terminals of the
two strands are at opposite ends where the two strands is antiparallel. The two
polynucleotide strands in the double helix DNA molecule are held together by hydrogen
bonds between complementary purine-pyrimidine base pairs. There are four different
types of nucleotides found in DNA, differing only in the nitrogenous which are adenine,
guanine, cytosine and thymine.

The sequence of steps in DNA replication is based on the semiconservative model

that is takes place in the 5’-3’ direction using a DNA template strand. This process is
catalyzed by DNA polymerase III. This process begin with the unwinding of the DNA
double helix to form a replication fork. The unwinding is catalyzed by the enzyme, DNA
helicase. The DNA helicase enzyme unwinds the double-stranded DNA to a replication
fork ready for replication. The antiparallel nature of the strands, that is the 5’-3’
orientation of the top strand and the 3’-5’ orientation of the complementary bottom
strand. Then, the DNA is already partially unwound to form a replication fork. On the
bottom template strand, RNA primase attaches a short RNA primer in the 5’-3’ direction.
After that, the RNA primase is removed, and free DNA nucleosides are added by DNA
poltmerase III to the RM|NA primer in the 5’-3’ direction. This new strand is called the
leading strand because it is made in the same direction as the movement of the replication
fork. On the top template strand, RNA primase synthesizes a short RNA primer in the a
short RNA primer in the 5’-3’ direction. RNA primase is removed, and DNA polymerase
III adds nucleosides to the RNA primer one after another opposite to the replication fork
to form a short length DNA. This new short length is called the lagging strand because it
is made in the direction opposite to the movement of the replication fork. The fragment
produced is also called Okazaki fragment. The process repeat as the DNA continues to
unwind. Then, the RNA primers are removed by a different type of enzyme called DNA
polymerase I. The two Okazaki fragments are then sealed and joined up by the DNA
ligase to produce a continuous chain called lagging strand antiparallel to the leading
strand formed earlier. Two new antiparallel continuous strands which are one leading
strand and one lagging strand, are formed. The new double-stranded daughter DNA
molecules daughter DNA molecules then twist or wind to form double helix DNA
molecules.

2.(a) Describe the steps in the synthesis of the polypeptide.

There are two processes that undergoes by a cell in the synthesis of the
polypeptide which are transcription and translation. Transcription is a process where the
information in DNA is transcribed to a RNA molecule, called messenger RNA (mRNA).
During transcription, one of the DNA strands acts as a template whereby a mRNA is
transcribed complementary to the DNA template strand. A specific protein-coding gene
consists of a promoter followed by the RNA-coding sequences for a protein and then a
terminator. The promoter is a base-sequence that specifies where transcription is begins
while the RNA-coding sequences is a base-pair sequence that includes coding
information for the polypeptide chain specified by the gene. Besides, the terminator is a
sequence that specifies the end of the mRNA transcript. Transcription is catalyzed by an
enzyme called RNA polymerase when initiation of the transcription at the promoter site
is begins. This involves specific recognition of promoter base sequence by RNA
polymerase (in prokaryotes) or a complex of proteins (in eukaryotes). RNA synthesized
is initiated with the addition of free RNA nucleotides in the 5’-3’ direction opposite with
attachment at -OH end to the uncoiling direction of the DNA segment. The same rule of
complementary base pairing are followed as in replication, except that uracil (U) replaces
thymine (T) to pair with adenine (A), as RNA does not contain tnymine. After the RNA
synthesis is initiated, the RNA elongation will continue to the direction of 5’-3’ direction
with the addition of more free nucleotides by RNA polymerase. Then, transcription will
terminate when the terminator base sequence is recognized by RNA polymerase. In
eukaryotes, introns and exons formed in the pre-mRNA during transcription are removed
to produce mature mRNAs.

While translation is a process where the genetic information transferred to a

mRNA from the DNA is translated by ribosomes into amino acid sequence on
polypeptides. This process requires the assistance of tRNA including the mRNA, rRNA
and ribosome, enzymes and ATP which are initiated with the movement of mRNA from
nucleus to ribosome. Ribosomes, the organelles on which the mRNA is translated,
consist of two subunits, each of which contains rRNA an ribosomal protein. Ribosomes
do not carry genetic information but facilitate the interaction of tRNA with mRNA during
protein synthesis. The small subunit of a ribosome, binds to the 5’ end of an mRNA
molecule. The small subunit slides along the mRNA until it reaches the start codon,
which shows where translation should be initiated. The large subunit then binds to the
small subunit to form a complete RNA-ribosome complex. The first tRNA occupies the
P site and the A site is available for the next tRNA. At the end of the initiation stage,
elongation begins when the vacant A site is bound by another charged tRNA with an
anticodon complementary to the codon after the start codon on the mRNA. The ribosome
moves three (a codon) nucleotides along the mRNA in a ‘5-3’ direction and detaches the
peptide from the tRNA at the P site. The detached peptide is then attached by a peptide
linkage to the single amino acid on the A site. In the process during which the ribosome
slides across to the next codon of the mRNA, the tRNA at the P site is then displaced and
detached from the ribosome. The P site is then adjacent tRNA carrying the polypeptide
chain. The vacant A site will the be occupied by the next tRNA. The steps repeat in a
cycle called ribosome until translation is terminated. When the ribosome reaches a stop
codon, there is no tRNA that has a complementary anticodon. This signals the end of
translation. The large subunit advances over the small subunit. The polypeptide is
detached from the tRNA, and starts to fold up to form the final shape of the protein. The
rRNA disintegrates to release the large subunit, small subunit, tRNA and mRNA.
(b) What would be the effect of a deletion or an addition in one of the DNA nucleotides

Base deletion is the loss or removal of one or more nucleotides from a DNA
nucleotide sequence while base addition is the insertion or addition of nucleotides into a
DNA nucleotide sequence. When a deletion or an addition in one of the DNA
nucleotides, it may alter a large portion of the resulting polypeptide. Base addition and
deletion are also known as frameshift mutation. A frameshift mutation is a gene mutation
that inserts or deletes a number of nucleotides (that are multiples of threes) from DNA
nucleotide sequence. For example, the sequence given, TAC-GAA-CTT-CGG-TCC is
inserted with a nucleotide base A and becomes TAA-CGA-ACT-TCG-GTC-C. This
insertion or deletion can disrupt the reading, or the grouping of the codons where it can
cause the codons to be read differently. Therefore, codons after the mutation will code
for different amino acids. Furthermore, the stop codons (UAA, UGA, and UAG) will not
be read, or a stop codon may be created at an earlier site. The protein created may be
abnormally short, abnormally long and contain the wrong amino acids. It will be most
likely not functional.

(c) What would be the effects of a substitution on one of the nucleotides?

Base substitution ocuur when one nucleotide is exchanged with another

nucleotide that has a different base. For example, TAC is exchanged with TAA. This
mutation alters only one amino acid. This can lead for different DNA sequence, which
will be transcribed into a different mRNA. Later, the altered mRNA will be translated
into a polypeptide with a different amino acid sequence from the normal one. This
mutation is also known as a missense mutation which mostly codes for a different amino
acod. The most common example of a base substitution effect is sickle-cell anemia.
3. Explain how the lac operon was controlled in prokaryotes

In eukaryotes, each gene is regulated separately. However, in prokaryotes such as

bacteria, some genes are arranged in clusters and are regulated together. This
clusters of gene are called operon, for example, the lactose operon found in E.
coli. The lac operon is composed of three lac genes (lacA, lacY and lacZ) coding
for different proteins, plus a promoter gene and an operator gene. A regulator
gene nearby codes for a repressor protein that binds to the operator when lactose
concentrations are low and effectively blocks RNA polymerase's access to the
promoter. This can cause the transcription is blocked. When milk is consumed
and lactose levels are high, the lactose binds to the repressor changing its shape
and effectively removing its blockage of the promoter. Then, RNA polymerase
can initiate transcription of the genes.

4. Describe the PCR process briefly.

PCR or Polymerase Chain Reaction is a method to produce many copies of

specific fragments of DNA in vitro. It resembles replication but is carried in a test tube.
The tools required for PCR include a thermocycler, a test tube containing the DNA
sample, the enzyme DNA polymerase, that is, Taq polymerase, primers and free DNA
nucleotides. The PCR is done from the original small sample of DNA fragment (target
DNA). Then, the DNA fragment is heated to break hydrogen bonds and separate the
strands. Each fragment is then cooled and the primers is added, so that they will bind to
the ends of the DNA. After that, the free DNA nucleotides are added nd the temperature
is raised. When the replication is completed to give two new complementary DNA
strands. Then, the processes are kept repeating to produce millions of copies of the target
DNA.
 TBB 1013

TUTORIAL CELL BIOLOGY

NAME : SITI AISHAH BINTI ABDUL LATIFF

MATRIC NO. : D 20081032348

I/C NO. : 890409025746

LECTURER’S NAME : PN. NORHAIDA