Gene Expresssion

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