BIOL2165 Tutorial 5- Molecular Genetics

1. Differentiate between theta and sigma mode of replication.

These are types of replication in bacteriophages. (1 mark)

The theta mode of replication begins at an origin by formation of a replication bubble proceeds
bidirectionally (1 mark) as a growing fork extending continuously on the leading strand and
discontinuously on the lagging strand (1 mark). Replication is semi-conservative (1 mark).
The sigma mode begins at a nick site at the outer +ve strand (1 mark). dNTPs are added to the 3’
end unidirectionally as the 5’ end is dragged out into a concatemer (1 mark). The –ve strand is
synthesized and resolved into unit lengths which circularise to form double stranded molecules
(1 mark).
Differences (4 marks)
Theta mode Sigma mode
Slow replication rate Fast replication rate
High fidelity Low fidelity (error rate 1000 x theta
mode
Growing fork model of Rolling circle mode of replication
replication
Uses host DNA polymerases Uses phage polymerase

34.

The following is a diagram of a gene and its regulatory sequences.

a. Label the components marked a-g in the above diagram.
a. Enhancer b. Promoter
c. TATA box (basal element) d. 5´ untranslated region (5´UTR).
e. Coding region f. Exons
g. 3´ untranslated region (3´UTR)
b. Is the above diagram a gene from a prokaryote or a eukaryote (1 mark)? Give TWO
reasons for your answer. (Any 2 reasons)
1. Only eukaryotic genes have introns.
2. Only eukaryotic genes have basal elements.
3. Only eukaryotic genes have enhancer sequences.
4. Only in eukaryotic genes are the promoters so dispersed; promoters of prokaryotic genes are
dispersed.

c. Which of the following sequences would be reflected in the:

i. Heterogeneous mRNA: d, e, g

ii. Mature mRNA: d, f, g

iii. Translated polypeptide: f

d. Name the process by which a heterogeneous mRNA is converted to a mature mRNA.

Post transcriptional modification

e. What changes take place during the process that you described in (d)?

• splicing introns
• Capping – addition of a modified guanosine at the 5’ end in an atypical 5’-5’ linkage. This
helps mRNA bind to the ribosome.
• Addition of a poly-A tail at the 3´end (200 nt long). This improves mRNA stability.

f. What are the functions of the sequences labeled d and g?

d: is the transcription start site, contains the Shine-Dalgarno sequence, targets mRNA to
ribosomes.

g: is the transcription stop site, protect mRNA ends.

g. What is the function of sequences labeled a and b?

a: Helps in transcription initiation, determines the rate of transcription.

b: Has conserved sequences which have homology to the sigma factor of RNA polymerase,
hence is where RNA polymerase binds and initiates transcription.

h. List in point form TWO roles of introns in eukaryotic genes. ( any 2)

1. Remnants of evolution
Introns are remnants of an ancestral approach to evolution of genes called ‘exon shuffling’
Therefore they are non-functional.
2. Developmental flexibility
One gene may give different polypeptides through:
• different start sites used in transcription
• different terminators used in transcription or
• alternate splicing pathways leading to different polypeptides.

3. Regulatory role
• in monocots the first intron has been shown to upregulate the transcription of the gene
• r2s oncogene- mutation in the intron has been shown to upregulate gene expression

4. Genome stability
• Introns are believed to provide greater stability to the genome by preventing mutations arising
due to imprecise recombination.
• The length of the introns is said to aid in recombination.

1. Describe the process of transcription in prokaryotes. Discuss the transcriptional control

mechanisms in prokaryotes using regulation of the lac operon as an example. (45 marks)
Transcription refers to the synthesis of a mRNA molecule by the DNA dependent RNA
polymerase enzyme according to the dictations of the genetic code on the sense stand of the double
helix. Hence transcription is asymmetric. (2 marks)

The E. coli the same RNA polymerase holoenzyme transcribes rRNA, tRNA and mRNA
genes. The core enzyme has the polymerase activity, while the sigma factor provides specificity
of recognition of promoters. (3 marks)

Coordinately expressed genes in prokaryotes are organized into operons and are transcribed
together to produce a polycistronic mRNA, which is later translated and resolved into individual
proteins. (3 marks)

Steps in Transcription:

1. Promoter recognition: The sigma factor of the RNA polymerase enzyme is involved in
recognition of promoters and is hence responsible for carrying the core enzyme to the
promoters. Promoters are upstream regulatory sequences and are involved in the regulation of
transcription rate. (2 marks)

2. Chain initiation: The binding of the RNA polymerase to the promoter causes localized melting
of a 10 bp region forming an open promoter complex. The first ribonucleotide is added at the +1
site, either ATP or GTP. Then the RBS is added before the translation start site AUG. The core
enzyme is involved in the polymerase activity. (3 marks)

3. Chain elongation: Transcription of the 3'--5' strand occurs in the 5'--3' direction. Once the RNA
polymerase moves 50-60 nucleotides downstream another RNA polymerase can initiate
transcription. (3 marks)

4. Chain termination: Termination occurs by the formation of secondary structures or at sites to

which rho proteins attach. Both of these can cause the RNA polymerase to slip off the transcribing
stand. (2 marks)

TRANSCRIPTIONAL CONTROL MECHANISMS IN PROKARYOTES (6 marks)

Transcription initiation
Positive control
Negative control
- Inducible system
- Repressible system
Transcription termination
Attenuation

Regulation of Lac Operon

The mechanism: The lac operon has a negative inducible system of control with a superimposed
positive control. Hence, the lac operon can function only in the absence of glucose and in the
presence of lactose. (4 marks)

Controlling elements: The Lac operon has the following organization (See fig) with two types of
controlling elements. The cis-acting elements are the promoter and the operator, which are
found immediately upstream of the structural genes and need to be on the same strand &
immediately upstream to be able to effect control. The regulatory gene (I) is a trans-acting element
and can function distal to the gene or from another DNA molecule. The I gene effects its control
on the Lac operon through a mobile repressor protein. (5 marks)
The negative control (inducible system) - The I gene produces a repressor protein which binds
to the operator region thus preventing the RNA polymerase from transcribing the gene. The Lac
operon is hence normally turned off and the repressor protein has to be removed to turn on the
gene. Hence negative control.

The repressor can be removed only in the presence of an inducer in the cell system - hence it is
inducible. Lactose serves as the inducer and in its presence, the lactose would bind to the repressor
protein, thus changing its allostery. This will result in greatly reduced affinity of the repressor to
the operator, causing the repressor to slip-off from the operator. Transcription can hence
continue. Hence, lactose-degrading enzymes are produced only in the presence of lactose. When
lactose is absent the Lac operon is shut off. (6 marks)

The positive control - The mechanism is called catabolic repression. When glucose is absent in
the cell, glucose catabolic products decrease. cAMP is a sensor molecule and increases in
response to decreases in glucose catabolites. cAMP binds to a protein called the Catabolite
Activator Protein (CAP) produced by the crp gene, and forms a CAP-cAMP complex. This
serves as an activator molecule to the Lac Operon. The activator needs to bind to upstream
elements of the promoter of the lac operon to allow the formation of the open promoter complex,
which is necessary for transcription to begin. Hence in the presence of glucose there are no
cAMP-CAP activator molecules and hence the lac operon cannot be turned on. Since the
activator molecules are involved in turning on the gene it is called positive control. (6 marks)