2. Outline
1. Gene expression in prokaryotic cells –
DNA to mRNA to protein.
2. Gene expression in eukaryotic cellsIntron splicing, 5’ capping, 3’-poly-A tail
3. DNA Replication
4. Reverse transcription
3. Every cell has the same DNA and therefore the same genes. But
different genes need to be “on” and “off” in different types of cells.
Therefore, gene expression must be regulated.
4. Gene expression must be regulated in
several different dimensions—
In time:
6 mos
14 wks
1 day
12 mos
10 wks
18 mos
At different stages of the life cycle, different genes need to be on and off.
6. 1. Gene expression : DNA to mRNA to
protein
• Gene expression – process where the information in a gene is
read and used to synthesize a protein
• Genetic information is linearly transferred from DNA to protein.
• What proteins you can make depends on what genes you have
Gene expression in prokaryotes
7. Transcription
• a messenger RNA (mRNA) molecule is synthesize using
the antisense strand as a template
• the genetic information is now transferred to the mRNA
• RNA is like DNA except : ribose sugar, single stranded,
uracil
9. Translation
• the information in the mRNA is read in a set of 3 bases – a
codon
• each codon codes for an amino acid
• a chain of amino acids – a polypeptide – is built by reading the
codons
• all these happen in the ribosome in the cytoplasm
11. The story is much more complicated in Eukaryotes
12. Important
differences
• A ‘cap’ is added to
the 5’ end of the
mRNA
• A polyA tail is
added to the 3’end
• Introns are
removed by a
process called
splicing
13. Introns and mRNA splicing
• Most eukaryotic genes
contain introns and exons
• Exons are DNA sequences
that carry genetic
information
• Introns do not carry genetic
information
• the introns are removed
from the mRNA by a
process called splicing
• whereby the introns are cut
out – and the exons are
rejoined
• this mature mRNA is then
translated to make proteins
15. 3. DNA replication
Every new cell must have a complete set of genes
Before cell division occurs, the DNA is replicated so that each
new cell has its own set of DNA
16. Overview
Synthesis of
the leading
strand
during
DNA
replication
Origin of replication
Leading strand
Lagging strand
Primer
Lagging strand
Leading strand
Overall directions
of replication
Origin of replication
3
5
RNA primer
5
“Sliding clamp”
3
5
Parental DNA
DNA poll III
3
5
5
3
5
17. In general :
• the original DNA molecules to serve as a template
• the new DNA strand is synthesized by the enzyme DNA
polymerase III
• Complementary base pairing ensures that the sequence of the
template is copied accurately
3‟
T
T
T
5‟
DNA polymerase III
New DNA strand
18. Synthesis of new DNA strand requires a primer
and can proceed only in a 5’ 3’ direction (why?)
5’ PO4
In the cell, the primer is a short RNA molecule
In the test tube, a short piece of DNA will also work.
19. DNA replication step-by-step
1) Double helix structure opened up by a helicase enzyme
The single stranded regions are stabilized by SSBs (single
stranded binding proteins)
22. DNA replication step-by-step
Direction of
replicasome
4) All these enzymes work together in a complex known as a
replicasome.
The replicasome moves in one direction, following the replication
fork
23. The two strands of a DNA are not equal
(when it comes to replication)
Replication can only happen
in a 5‟ to 3‟ direction
„leading‟ and „lagging‟
strands
24. DNA replication step-by-step
On the leading strand, everything’s OK
- DNA synthesis occurs continuously in a 5’ 3’ direction
25. DNA replication step-by-step
On the lagging strand, we have a problem
- DNA synthesis cannot happen in a 3’ 5’ direction
- thus, multiple primers are made
- new DNA is synthesized as small Okazaki fragments (5’ 3’)
- the primers are then replaced with DNA by DNA polymerase I
- the DNA fragments are then joined by DNA ligase
26. Fig. 16-17
A summary of bacterial DNA replication
Overview
Origin of replication
Lagging strand
Leading strand
Leading strand
Lagging strand
Overall directions
of replication
Single-strand
binding protein
Helicase
5
3
Parental DNA
Leading strand
3
DNA pol III
Primer
5
Primase
3
DNA pol III
Lagging strand
5
4
DNA pol I
3 5
3
2
DNA ligase
1
3
5
27. 3
Synthesis of the
lagging strand
5
5
Template
strand
3
3
RNA primer
3
1
5
5
3
1
5
3
5
2
3
3
5
Okazaki
fragment
3
5
1
5
3
5
2
1
5
3
1
2
Overall direction of replication
3
5
28. Proof reading minimized replication error
DNA polymerase III has a 3‟ 5‟ exonuclease activity that can
cut and repair mistakes
Remember : DNA replication has to be very accurate (or else?)
29. DNA replication is semi conservative
Replication : From one DNA molecules to two
Identical sequences
32. The transfer of genetic information from RNA to DNA
• By the enzyme reverse transcriptase found in retrovirus
• This allows us to make cDNA (complementary DNA) from mRNA
• and obtain a gene sequence without the introns
Reverse transcription
cDNA
