Author of Lecture: Title of Lecture:

Clifton-Bligh, Rory (Dr) Genetic Medicine 2: Principles of molecular genetics

(Problem 1.03, Lecture 10, 2011)

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Principles of molecular biology

Overview
The

genetic paradigm Part 1: DNA and chromosomes Part 2: The language of heredity: from DNA into protein Part 3: Regulation of gene expression Part 4: where things go wrong

The Genetic Paradigm

parents
transposons

miRNAs

RNA
Reverse transcription

protein

function

children

The Genetic Paradigm

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 Mantra

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

The Genetic Mantra

Inheritance: Chromosomal eg Turners syndrome

parents

Mendelian

RNA
Mosaic

protein

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

function

Mitochondrial - eg diabetes-deafness - eg McCune-Albright - eg

pseudohypoparathyroidism

children

Imprinting

Uniparental disomy Multigenic

The Genetic Mantra

parents RNA children protein function

Chemical nature of DNA

Nucleic acids contain

Compacted in several stages

Four different nitrogenous bases ATGC Pyrimidines CT Purines AG Pentose sugar (deoxyribose) phosphate DNA+histones = nucleosomes Nucleosomes+non-histone proteins = chromatin Euchromatin = actively transcribed Heterochromatin = inactive

Chemical nature of DNA

Thymidine
HC N C CH3 C C N O H N H C N HC H O H N H N H C N C C CH C N N N C CH C N N H

P
HC N

O H C C N C O H N

Adenine

Cytosine

Guanine

Chemical nature of DNA

T Thymidine

CTAGGTCATTAAGGCCACGTGCAT A Adenine GATCCAGTAATTCCGGTGCACGTA

C Cytosine

Guanine G

Organisation of DNA

CTAGGTCATTAAGGCCACGTGCAT GATCCAGTAATTCCGGTGCACGTA

The double-helix is the basis of heredity

Explains: Replication

Inheritance Complementary basepairing also central to understanding RNA, protein

Organisation of DNA

HISTONES

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

What is a gene?

The Gene

Region of DNA transcribed into RNA Unit of heredity Humans: 30 000-40 000 genes Expressed regions (exons) ie present in mature mRNA Intervening regions (introns) are excised as a new RNA transcript is processed to its mature form Promoter: initiates transcription and regulates efficiency of transcription

The language of the genome

Translation

RNA

protein

function

Transcription

Post-translation

Transcription
Recognition

of the promoter
START

Regulatory elements

core promoter TATA box

Transcription
Recognition
IIA

of the promoter
IIF IIB IIE

Transcription factors IID Pol II IIH

Transcription
RNA

polymerase transcribes RNA from DNA

Pol II

..CAUG.. ..GTAC..

Transcription
RNA

transcript undergoes modification

CAP

AAAAAAAAAAAAAAn Pol II

Transcription
Splicing

exons (introns removed)

Introns

CAP

AAAAAAAAAAAAAAn

Exons

Transcription
Messenger

RNA is exported from the nucleus to cytoplasm

CAP

AAAAAAAAAAAAAAn

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

RNA codons

GGCCGCAUGAACCGUCGCCCGUCACCGUUAUUGCGU
methionine asparagine arginine arginine proline serine proline leucine

The Genetic Code

Redundant ie several nucleotide triplets U may code for one amino acid C Universal
A G

U
Phe Leu Leu Ile Met Val

C
Ser

A
Tyr
stop stop

G
Cys Trp Arg Ser Arg Gly
stop
U C A G U C A G U C A G U C A G

Pro

His Gln Asn Lys Asp Glu

Thr

Ala

Translation
The

ribosome:

Large ribosomal subunit

Small ribosomal subunit

Transfer

RNA
Amino acid

Anti-codon

C
Ser

A
Tyr
stop stop

G
Cys Trp Arg Ser Arg Gly
stop
U C A G U C A G U C A G G U C A G

Translation
methionine

U C A G

Phe Leu Leu Ile Met Val

Pro

His Gln Asn Lys Asp Glu

Thr

UAC

Ala

AUG

mRNA

Translation

UAC AUG

Translation

Growing polypeptide chain

Translation

U U C A G
Phe Leu Leu Ile Met Val

C
Ser

A
Tyr
stop stop stop

G
Cys Trp Arg Ser Arg Gly
stop

Post-translational modification
U C A G U C A G U C A G UAG

U C A G G

Pro

His Gln Asn Lys Asp Glu

Thr

Ala

Protein modification

The shape of a protein is specified by amino acid sequence Molecular chaperones

Hsp60 and hsp70 Prevent protein aggregation Fold protein into correct 3-d form

Quality control: ubiquitination Post-translational modifications

Disulfide bonds Glycosylation phosphorylation

Protein folding

Kuznetsov, G. et al. N Engl J Med 1998;339:1688-1695

Post-translation modification
Preproinsulin Proinsulin

insulin

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

The basic idea

RNA
Which protein How much

protein

function

When (timing)

In what combination

Regulation of gene expression

For most genes the most important control point of expression is at the initiation of RNA transcription

DNA level: chromatin, histones, promoters, enhancers RNA level: RNA stability Protein level: protein degradation, modification Amount of a gene expressed Type of gene product expressed

Chromatin structure and transcription

Ac Histone complex Ac Histones acetylated =transcription facilitated

Histones deacetylated =transcription repressed

Transcription factors can recruit histone modifying enzymes

Histone Deacetylase

switch

Histone Acetylases

Gene regulation by DNA

Nuclear or Receptor

Transcription Factor

Nuclear receptor

Histone acetylases

IIAIIB IIF IID

IIE

Pol II IIH

Other Transcription factors

Gene regulation at RNA level

Differential splicing can produce

Variant of same protein product

Different protein product

E1 E1 E2 E2 E3 E3
calcitonin CGRP

gene RNA

E6 E6

calcitonin

CGRP

E1

E2 E3 calcitonin

E1

E2 E3

CGRP

E6

calcitonin
Guttmacher, A. E. et al. N Engl J Med 2002;347:1512-1520

CGRP

Summary: part 3

Control of gene expression is mostly at the point of transcription Gene transcription is controlled by combinations of regulatory proteins Each cell type has a specific combination of regulatory proteins that directs gene expression appropriate to that cell Histone modification affects gene expression

Coding DNA mutations

Silent Missense Nonsense Frameshifts

Coding DNA mutations

Silent

Normal sequence: CCG TCA CCG

Serine Silent mutation: CCG TCC CCG

Serine

Coding DNA mutations

Silent Missense

Normal sequence: CCG TCA CCG

Serine Missense mutation: CCG CCA CCG

Proline

Coding DNA mutations

Silent Missense

Normal sequence: CCG TCA CCG

Serine

Nonsense

Missense mutation: CCG TAA CCG

Stop

Coding DNA mutations

Silent Missense

Normal sequence: CCG TCA CCG

Serine Proline

Nonsense Frameshifts

Frameshift mutation: CCG TCG ACC G

Serine Threonine

Coding DNA mutations

Can

reduce amount of mRNA (and therefore protein) alter function of protein (eg mutation in catalytic site of enzyme either activating or inactivating) cause protein mis-folding

Can

Can

Cystic fibrosis: mutations in CFTR gene

Defective protein production usually caused by nonsense, frameshift, or splice-site mutations, leading to premature termination of the mRNA and complete absence of CFTR protein. Defective protein processing these mutations prevent the protein from trafficking to the correct cellular location. Includes the most common CFTR mutation, delta F508 (deletion of a phenylalanine residue at codon 508). Defective channel function These mutations lead to diminished channel activity or rate of ion flow when compared to normal CFTR.

UpToDate, 2004

Suggested reading

Molecular Biology of the Cell by Alberts, Johnson, Lewis, Raff, Roberts, Walter. Garland Publishing, 2002. Genes VII by Benjamin Lewin, Paul Siliciano, Martin Klotz. Oxford University Press, 1999. A Genetic Switch: Phage and Higher Organisms by Mark Ptashne. Blackwell Science, Inc., 1992.

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

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. 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

Different gene expressed

Cystic fibrosis: severity correlates with amount of functional CFTR

Different mutations variably affect CFTR expression and function

Stern, R. C. N Engl J Med 1997;336:487-491

Protein misfolding
Aggregations

of misfolded protein can cause disease:

Cystic fibrosis 1- antitrypsin deficiency Huntingtons disease Alzheimers disease Parkinsons disease (familial) Amyloid Prion diseases

Carrell and Lomas 2002. Alpha1-Antitrypsin deficiency- A Model for Conformational Diseases. New Engl J Med; 346:45-53

Mutations in different regions of the gene can produce different phenotypes

Burke, W. N Engl J Med 2003;349:969-974

A special kind of insertion

Trinucleotide repeats Found in several genes Number varies among healthy individuals Increase in number of repeats above a critical threshold associated with disease Eg Huntingtons disease

Chromosomal translocations can produce new genes

Translocation (9,22) Leading to the Philadelphia (Ph) Chromosome and the Role of BCR-ABL in the Pathogenesis of CML

Savage, D. G. et al. N Engl J Med 2002;346:683-693

Splice-site mutations
Loss

of critical exon gene expression

Reduced Eg

several common forms of alpha and beta thalassemia