molecular basis.pdf

10/18/0910/18/09

Principle of Molecular BiologyPrinciple of Molecular Biology

Dr.Mohammed AlbalwiSection 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 g self-raising flour

1 teaspoon ground ginger

cream

Sometimes a mistake in the recipe doesn’t matter…

Gingerbread balls

100 g butter

350 g caster sugar

2 egg

250 g self-raising flour

1 teaspoon ginger

cream

And sometimes a mistake creates a disaster…

Gingerbread balls

100 g butter

350 g caster sugar

1 egg

250 g self-raising flour

1 teaspoon Vinegar

cream

Outline

The Genetic DogmaThe Genetic DogmaPart 1: Part 1: DNA and Chromosome Structure

Part 2: The Language of the heredity: from The Language of the heredity: from DNA to ProteinDNA to ProteinPart 3: Regulation of Gene Expression Regulation of Gene Expression MutationsMutationsPart 4: Part 4: Where things goes wrong?Where things goes wrong?

The Genetic DogmaThe Genetic Dogma

DNADNA RNA protein function

parents

children

Reverse transcription

tRNAsSINEssiRNAs

The Genetic DogmaThe 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 DogmaThe Genetic Dogma

Disease occurs in the presence of an environmental insult and a genetically susceptible individual

The Genetic MantraThe Genetic Mantra

RNA protein function

parents

children

DNADNA

Inheritance:Chromosomal – eg Turners syndrome

Mendelian – Autosomal dominant- Autosomal recessive- X-linked- Y-linked

Mitochondrial - eg diabetes-deafness

Mosaic - eg McCune-Albright

Imprinting - eg Prader-Willi Syndrome

Uniparental disomy

Multigenic

What molecular substance is a What molecular substance is a ““genegene”” made of?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

3’ end

5’ end 3’ end

• Long chain of polymer

(polynucleotide)

• The basic shape is like a twisted ladder or zipper (double helix).

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

Euchromatin

Hetrochromatin

Nucleolus

q arm

centromer

Nucleus

What IS the genetic material feature? What IS the genetic material feature?

2. Able to 2. Able to store informationstore information

3. Stable so that it 3. Stable so that it can be copied and passed oncan be copied and passed onthrough generationthrough generation

1. Lies on chromosome1. Lies on chromosome

4. Able to 4. Able to undergo rare changesundergo rare changes called mutations called mutations in order for evolution to occurin 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 GeneticThe Central Dogma of Molecular Genetic

Replication

Transcript TranslationDNA RNA Protein

phenotypeGenotype

III

DNA replication (DNA synthesis)

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 & Gene Definition & AnatomyAnatomy

Gene Structure

ATG (Initiation site) TAG or TAA or TGA

Regulatory region

TranscriptionTranscription

• Recognition of the promoter

START

Regulatory elements “core” promoterTATA box

Transcription

Recognition of the promoter

IIA

IID

IIB

IIFIIE

Pol II IIH

Transcription factors

Transcription (RNA synthesis)

II

Promoter

Transcription

RNA polymerase transcribes RNA from DNA

Pol II

..CAUG..

..GTAC..

1. Initiation rNTP (ATP, GTP, CTP & UTP)

Transcription (RNA synthesis)

2. Elongation

RNA product

3. Termination

RNA Processing Heterogeneous nuclear RNAhnRNA

Large Primary Transcript RNA(Pre-RNA)

CAP and Tailing added

7-Methyl G (Guanosine) Poly A Tail

(AAAAAA)n

Introns Splicing Out

AUG UAA

Mature RNA

5’ UTR UTR ‘3

Transcription

RNA transcript undergoes modification

Pol II

AAAAAAAAAAAAAAnCAP

TranscriptionTranscription

• Splicing exons (introns removed)

Exons

Introns

AAAAAAAAAAAAAAnCAP

TranscriptionTranscription

• Messenger RNA is exported from the nucleus to cytoplasm

AAAAAAAAAAAAAAnCAP

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'

•Polypeptides are built on the ribosome on a polysome

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

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

Only 20 Amino Acids

The Genetic CodeThe Genetic Code

• Redundanti.e. several nucleotide triplets may code for one amino acid

• Universal

TranslationTranslation

Large ribosomal subunit

• The ribosome:

• Transfer RNA

Small ribosomal subunit

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 tRNAwill be joined to the mRNA-ribosome complex.

A: amino acid

P: peptide

E: exit

Translation Steps

3. ELONGATION

Adding new aa's (Peptidyltransferase)

Translation Steps4.TERMINATION stopping the process

Translation

AUG

UAC

methionine

U C A G

U

C

A

G

UCAG

UCAG

UCAG

UCAG

PheLeu

SerTyr

Leu ProHisGln

Arg

IleMet

ThrAsnLys

SerArg

Val AlaAspGlu

Gly

Cys

Trpstopstop

stop

A

U

GMet

mRNA

Translation

AUG

UAC

Translation

Growing polypeptide chain

Translation

U C A G

U

C

A

G

UCAG

UCAG

UCAG

UCAG

PheLeu

SerTyr

Leu ProHisGln

Arg

IleMet

ThrAsnLys

SerArg

Val AlaAspGlu

Gly

Cys

Trpstopstop

stopU

A

Gstop

UAG

Post-translational modification

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

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 codonsand cognate amino acids

• Proteins are folded and processed to achieve function

Differences between bacterial and Differences between bacterial and eukaryotic eukaryotic ribosomesribosomes can be can be

exploitedexploitedInhibitors of bacterial protein synthesis are used as antibiotics

• Tetracycline• Streptomycin block• Chloramphenicol ribosome• Erythromycin function

• Rifampicin: blocks RNA polymerase

The basic ideaThe basic idea……

RNA protein functionDNADNA

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

Histones acetylated=transcription facilitated

Histones deacetylated=transcription repressed

Histonecomplex

Chromatin structure and transcription

HistoneDeacetylase

“switch”

HistoneAcetylases

2. Post-Transcriptional Regulation

RNA level: RNA stability

3. Transltional Regulation

-- Availability of Availability of tRNAtRNA, amino acids, etc., amino acids, etc.

-- Shelf Life of mRNAShelf Life of mRNA

-- HormonesHormones--affect stabilityaffect stability

- Activated proteins- enzyme cleavage* Removal of "signal" peptide sequences

* Addition of sugar residues (glycosylation)

* Phosphorylation etc

- Degradation of proteins

4. Post-Transltional Regulation

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) DNAmolecules into recipient cell

Problems that occur

Dysregulated gene expression•DNA Methylation•DNA mutations:

PromoterCoding regionSplice-site

•Non-DNA changesHistone modification

MethylationMethylation

• Methylation of cytosines=silencing• Eg X chromosome inactivation• Important cause of imprinting• Too much or too little methylation can

cause imprinted disordersBeckwidth-Wideman syndromeAngelman syndrome

DNA MutationsDNA 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 mutationsCoding 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!Thanks for your attention!Any questions?Any questions?