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10/18/09

Principle of Molecular Biology


Dr.Mohammed Albalwi
Section of Molecular Pathology and Genetics
Department of Pathology and Laboratory Medicine
Balwim@ngha.med.sa
Ex.11287 / pager 1287

Molecular biology is a lot like cooking


Gingerbread balls
{100

g butter

{350

g caster sugar

{1

egg

{250
{1
{

g self-raising flour

teaspoon ground ginger

cream

Sometimes a mistake in the recipe doesnt


matter
Gingerbread balls
{100

g butter

{350

g caster sugar

{2

egg

{250
{1
{

g self-raising flour

teaspoon ginger

cream

And sometimes a mistake creates a


disaster
Gingerbread balls
{100

g butter

{350

g caster sugar

{1

egg

{250
{1
{

g self-raising flour

teaspoon Vinegar

cream

Outline
The Genetic Dogma
Part 1: DNA and Chromosome Structure
Part 2: The Language of the heredity: from
DNA to Protein
Part 3: Regulation of Gene Expression
Mutations
Part 4: Where things goes wrong?

The Genetic Dogma

tRNAs
SINEs
siRNAs

parents
DNA

RNA

Reverse transcription

children

protein

function

The Genetic Dogma


Nucleated cells from the same organism have
the same genome
Differences in gene expression determines
different cellular function
The whole organism is the product of
interaction between its genes and the
environment

The Genetic Dogma

Disease occurs in the presence of


an environmental insult and a
genetically susceptible individual

The Genetic Mantra


Inheritance:
Chromosomal eg Turners syndrome

parents
DNA

Mendelian

RNA

Autosomal dominant
- Autosomal recessive
- X-linked
- Y-linked

protein

function

Mitochondrial - eg diabetes-deafness

children

Mosaic

- eg McCune-Albright

Imprinting

- eg

Prader-Willi Syndrome

Uniparental disomy
Multigenic

What molecular substance is a gene made of?


A segment or specific sequence of a DNA molecule that
contains the information required for the synthesis of a
functional biological product, whether protein or RNA.

How Gene Work


One gene One protein? / two proteins?
One enzyme?
One functional biological product?
One specific sequence?
One unchangeable?
One disease or disorder?

DNA Structure

5 end
Long chain of polymer
(polynucleotide)
The basic shape is like a
twisted ladder or zipper
(double helix).

3 end

3 end

Types of RNA
1. mRNA (messenger): takes a message from the nucleus to
the ribosomes in the cytoplasm
2. tRNA (transfer): transfers amino acids to the ribosomes
3. rRNA (ribosomal): along with proteins, makes up the
ribosomes where polypeptides are synthesized.
4. smRNA, snoRNA and microRNA

DNA Organization
Helix

Nucleosomes

Solenoid

Chromomere
Chromatin

Chromosome

P arm
Nucleolus
Euchromatin
centromer
Hetrochromatin
q arm
Nucleus

What IS the genetic material feature?

1. Lies on chromosome
2. Able to store information
3. Stable so that it can be copied and passed on
through generation
4. Able to undergo rare changes called mutations
in order for evolution to occur

Summary: part 1
DNA consists of two complementary chains
of nucleotides
The double-helix is the mechanism for
heredity
DNA resides in the nucleus (eukaryotes)
DNA is packaged in chromosomes
Chromosomes contain long strings of
genes

The Central Dogma of Molecular Genetic


Replication

DNA
Genotype

Transcript

RNA

Translation

Protein
phenotype

DNA replication (DNA synthesis)

III

III

DNA replication (DNA synthesis)


Several enzymes are required

Helicase (Untwists DNA)


Single Strand Binding Proteins(Stabilize replication fork)
Primase (Makes RNA primer)
DNA polymerase III (Synthesizes new DNA strands)
DNA ligase repairs Okazaki fragments (seals lagging strand

holes)

Gene Definition &


Anatomy

Gene Structure
ATG (Initiation site)

Regulatory region

TAG or TAA or TGA

Transcription
Recognition of the promoter
START

Regulatory elements

core promoter
TATA box

Transcription
{

Recognition of the promoter


IIF
IIA

IIE

IIB

Transcription factors

IID

Pol II

IIH

Transcription (RNA synthesis)

Promoter
II

Transcription
{

RNA polymerase transcribes RNA


from DNA

Pol II

..CAUG..
..GTAC..

1. Initiation

Transcription (RNA synthesis)


rNTP (ATP, GTP, CTP & UTP)

2. Elongation

3. Termination
RNA product

RNA Processing

7-Methyl G (Guanosine)

Heterogeneous nuclear RNA


hnRNA

Large Primary Transcript RNA


(Pre-RNA)

Poly A Tail
(AAAAAA)n

CAP and Tailing added

Introns Splicing Out

AUG

UAA

Mature RNA
5 UTR

UTR 3

Transcription
{

CAP

RNA transcript undergoes


modification

AAAAAAAAAAAAAAn

Pol II

Transcription
Splicing exons (introns removed)
Introns

AAAAAAAAAAAAAAn

CAP

Exons

Transcription
Messenger RNA is exported from
the nucleus to cytoplasm

CAP

AAAAAAAAAAAAAAn

RNA Transportation

Summary of Transcription
Recognition of the promoter
RNA polymerase transcribes RNA from DNA
RNA transcript undergoes modification
Splicing exons (introns removed)
Messenger RNA is exported from the nucleus
to cytoplasm

Translation (Protein synthesis)


Process of making a protein in a specific amino acid sequence from
a unique mRNA sequence...
unit of information is CODON

genetic 'word'

3 nucleotides = 1 codon (word) = 1 amino acid

Polypeptides are built on the ribosome on a polysome

Genetic CODE 64 triplet codons [61 = AA & 3 stop codons]

Only 20 Amino Acids

The Genetic Code


Redundant
i.e. several
nucleotide triplets
may code for one
amino acid
Universal

Translation
The ribosome:

Large ribosomal subunit

Small ribosomal subunit

Transfer RNA

Amino acid

Anti-codon

Translation Steps
1. ACTIVATION
add an amino acid to tRNA

Translation Steps
2. INITIATION
Assemble players [ribosome,
mRNA, aa-tRNA]

A, P & E are sites where tRNA


will be joined to the mRNAribosome complex.
A: amino acid
P: peptide
E: exit

Translation Steps
3. ELONGATION

Adding new aa's (Peptidyl


transferase)

Translation Steps
4.TERMINATION

stopping the process

Translation
methionine

Leu

Ile
Met

UAC

AUG

mRNA

Phe
Leu

Val

C
Ser

Tyr

Cys

stop
stop

Trp

stop

U
C
A
G

Pro

His
Gln

Arg

U
C
A
G

Thr

Asn
Lys

Ser
Arg

U
C
A
G

Ala

Asp
Glu

Gly

U
C
A
G

Translation

UAC
AUG

Translation

Growing polypeptide chain

Translation

U
U

Phe
Leu

Leu

Ile
Met

Val

C
Ser

Tyr

Cys

stop
stop
stop

Trp

stop

U
Post-translational
modification
C
A
G

Pro

His
Gln

Arg

U
C
A
G

Thr

Asn
Lys

Ser
Arg

U
C
A
G

Ala

Asp
Glu

Gly

U
C
A
G

UAG

Summary of Translation
mRNA sequence is decoded in sets of three
nucleotides called codons
Each codon specifies an amino acid
Each mRNA therefore encodes a polypeptide chain
Although there are 3 possible reading frames
for mRNA, only one is used

Post-Translation Process

Hsp60
Hsp70

Post-translational modifications
z

Glycosylation

phosphorylation

Disulfide bonds

Summary: part 2
A gene is DNA that is transcribed into RNA
RNA is complementary to one DNA strand
RNA is modified before export from the
nucleus
mRNA sequence is decoded in triplet
nucleotides by ribosomes
tRNAs are the bridge between RNA codons
and cognate amino acids
Proteins are folded and processed to
achieve function

Differences between bacterial and


eukaryotic ribosomes can be
exploited
Inhibitors of bacterial protein
synthesis are used as antibiotics
Tetracycline
Streptomycin
block
Chloramphenicol
ribosome
Erythromycin
function
Rifampicin: blocks RNA
polymerase

The basic idea

DNA

RNA

protein

function

Which protein
How much
When (timing)

In what combination

Gene Regulation
Why genes are regulated?
It would be a waste of cellular energy and
materials if every gene was constantly being
actively transcribed.
Some genes are rarely used, and only in specific
cells.
Different genes need to be activated at different
times, depending on location in the body and
time.

Control Gene Expression


1. Transcriptional Regulation
DNA level: chromatin,
histones, promoters,
enhancers
RNA level: RNA stability

Chromatin structure and transcription

Ac

Ac
Histone
complex

Histones acetylated
=transcription facilitated

Histones deacetylated
=transcription repressed

Chromatin structure and transcription

Histone
Deacetylase

switch

Histone
Acetylases

2. Post-Transcriptional Regulation

RNA level: RNA


stability

3. Transltional Regulation
- Availability of tRNA, amino acids, etc.
- Shelf Life of mRNA
- Hormones-affect stability

4. Post-Transltional Regulation
- Activated proteins- enzyme cleavage
* Removal of "signal" peptide sequences
* Addition of sugar residues (glycosylation)
* Phosphorylation etc

- Degradation of proteins

GENETIC CHANGE
Change in DNA nucleotide sequence
Mutation

a change in DNA that results in a different codon =


different amino acid sequence
Recombination
change in genotype by insertion of NEW (foreign) DNA
molecules into recipient cell

Problems that occur


Dysregulated gene expression
DNA Methylation
DNA mutations:
Promoter
Coding region
Splice-site
Non-DNA changes
Histone modification

Methylation

Methylation of cytosines=silencing
Eg X chromosome inactivation
Important cause of imprinting
Too much or too little methylation can
cause imprinted disorders
Beckwidth-Wideman syndrome
Angelman syndrome

DNA Mutations
Promoter mutations
Reduce gene expression
Eg some cases of alpha and beta thalassemia
are due to mutations in promoter or enhancer
sequences for the alpha or beta globin genes.
Different gene expressed
Eg glucocorticoid-remediable hypertension is
due to fusion of the promoter region of the
gene for CYP11B1 and the coding sequences of
CYP11B2, resulting in ACTH-dependent
activation of the aldosterone synthase

Coding DNA mutations

Can reduce amount of mRNA (and therefore


protein)
Can alter function of protein (eg mutation in
catalytic site of enzyme either activating or
inactivating)
Can cause protein mis-folding

Mutations (Where?)
Somatic mutations
- happens in a non-germ line cell
- organism becomes a mosaic
- are the cause of some cancers
Germ-line mutations
- occurs in the cells that give rise to gametes
- gamete will carry the mutation to the next generation
- germ-line mutations are heritable

Mutation

silent

Thanks for your attention!

Any questions?