gene expresssion
TRANSCRIPT
MIC210 BASIC MOLECULAR BIOLOGY
By
SITI NORAZURA JAMAL (MISS AZURA) 03 006/06 483 2132
Lecture 3
Gene Expression
Outline
1. Gene expression in prokaryotic cells – DNA to mRNA to protein.
2. Gene expression in eukaryotic cells- Intron splicing, 5’ capping, 3’-poly-A tail
3. DNA Replication
4. Reverse transcription
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.
Gene expression must be regulated in several different dimensions—
In time: 10 wks 14 wks 1 day
6 mos 12 mos 18 mos
At different stages of the life cycle, different genes need to be on and off.
1) Gene expression in prokaryotic cells– DNA to mRNA
to protein.
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
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
Molecular Components of Transcription
• RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides
• RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine
• The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator
• The stretch of DNA that is transcribed is called a transcription unit
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
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
2) Gene expression in eukaryotic cells- Intron splicing, 5’ capping,
3’-poly-A tail
The story is much more complicated in Eukaryotes
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
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
3) DNA Replication
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
Leading strand
Overview
Origin of replication
Lagging strand
Leading strand Lagging strand
Primer
Overall directions of replication
Origin of replication
RNA primer
“Sliding clamp”
DNA poll III Parental DNA
5
3
3
3
3
5
5
5
5
5
Synthesis of
the leading
strand
during
DNA
replication
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
T T T
New DNA strand DNA polymerase III
5‟
3‟
Synthesis of new DNA strand requires a primer
and can proceed only in a 5’ 3’ direction (why?)
In the cell, the primer is a short RNA molecule
In the test tube, a short piece of DNA will also work.
5’ PO4
1) Double helix structure opened up by a helicase enzyme
The single stranded regions are stabilized by SSBs (single
stranded binding proteins)
DNA replication step-by-step
2) Another enzyme, primase, makes a short RNA
primer
DNA replication step-by-step
3) Then DNA polymerase III begins to extend the new DNA
strand
DNA replication step-by-step
4) All these enzymes work together in a complex known as a
replicasome.
The replicasome moves in one direction, following the replication
fork
DNA replication step-by-step
Direction of
replicasome
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
On the leading strand, everything’s OK
- DNA synthesis occurs continuously in a 5’ 3’ direction
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
DNA replication step-by-step
Fig. 16-17
Overview
Origin of replication
Leading strand
Leading strand
Lagging strand
Lagging strand Overall directions
of replication
Leading strand
Lagging strand
Helicase
Parental DNA
DNA pol III
Primer Primase
DNA ligase
DNA pol III
DNA pol I
Single-strand
binding protein
5
3
5
5
5
5
3
3
3
3 1 3
2
4
A summary of bacterial DNA replication
Template
strand
5
5 3
3
RNA primer 3 5
5
3
1
1
3
3
5
5
Okazaki
fragment
1 2
3
3
5
5
1 2
3
3
5
5
1 2
5
5
3
3
Overall direction of replication
Synthesis of the
lagging strand
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?)
DNA replication is semi conservative
Replication : From one DNA molecules to two
Identical sequences
4) Reverse Transcription
4. Reverse transcription – from RNA to DNA
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