Properties and functions of Properties and functions of nucleic acidsnucleic acids

These slides provides an overview of some of the properties of nucleic acids (DNA and RNA) and its applications in molecular biology

Dr. Momna Hejmadi, University of Bath

N.B. Some images used in these slides are from the textbooks listed and are not covered under the Creative Commons license as yet

N.B. Some images used in these slides are from the textbooks listed and are not covered under the Creative Commons license as yet

DNA basics resources created by Dr. Momna Hejmadi, University of Bath, 2010, is licensed under the Creative Commons Attribution-Non-Commercial-Share Alike 2.0 UK: England & Wales License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/2.0/uk/ or send a letter to Creative Commons, 171 Second Street, Suite 300, San Francisco, California 94105, USA.

learning objectives1) Compare sizes of DNA and understand the C-value

paradox

2) Understand the Human Genome Project

3) Be able to describe how the different helical topologies of DNA contribute to packing?

4) Understand the factors that contribute to the stability of the DNA double helix?

5) Appreciate the diverse functions of nucleic acids

ReferenceChapter 29: Biochemistry (3e) by D Voet and J Voet

(Wiley Publishing)

Genomes and the Human Genome project

C-value paradox

DNA topology and function

Factors that stabilise DNA

a) denaturation and renaturation

b) Sugar-phosphate chain conformations

c) Base pairing and base stacking

d) hydrophobic and ionic interactions

Functions of nucleic acids

OutlineOutline

DNA vs RNA sizeDNA molecules tend to be larger than RNA molecules

genome sizes

organism Number of base pairs (kb)

 

 

virusesLambda bacteriophage ( λ) 48.6

bacteriaEschericia coli 4,640

eukaryotesYeast 13,500Drosophila 165,000Human 3.3 x 106

What is the Human genome project?

Public consortiumHeaded by F CollinsStarted in mid 80’sWorking draft completed in 2001

Nature: Feb 2001

Celera GenomicsHeaded by C VenterStarted in mid 90’sWorking draft completed in 2001

Science: Feb 2001

Human genome = 3.3 X 10 9 base pairsNumber of genes = 26 – 32,000 genes

Goal: to sequence the entire human nuclear genome

The human genomeThe human genome

Nuclear genome (3.2 Gbp) 24 types of linear chromosomes Y- 51Mb and chr1 -279Mbp~ 30,000 genes

Mitochondrial genome (16.6kbp) – multicopy, circular, ds DNA

Gene and gene-relatedGene and gene-related

Everything else Everything else

Why do we need the DNA blueprint?

Individual human variation is 0.1%i.e. 1.4 million sequence variations

Applications in medicine, forensics, bioarchaeology, anthropology, human evolution, human migration etc

....in disease

……or risk of disease

N(291)S

....or in pharmacogenomics

what can a single human hair tell you?

nuclear DNAnuclear DNAHair rootHair root

mitochondrial DNAmitochondrial DNAHairHair shaftshaft

....or in forensics

Does size matter? C-value Does size matter? C-value paradoxparadox

mountain grasshopper Podisma pedestrisGenome size: 18 Gbp

protozoan Amoeba dubiaGenome size: 670Gbp

Boa constrictorGenome size: 2.1 Gbp

Homo sapiens sapiensGenome size: 3.2 Gbp

C value: DNA content of any haploid cellC value: DNA content of any haploid cell

CComparatiomparative genome ve genome sizessizes 

 

Why is there a discrepancy between genome size and genetic complexity?

C-value paradox

 

Genome sizes vary due to the presence of repetitive DNA

Repetitive DNA families constitute nearly one-half of genome (~45%)

Protein domains contribute to organism complexity

Explaining the paradox

Largest known mammalian gene is….DMD gene 2.5 Mbp (0.1% of the genome)

Mutations cause Duchenne’s muscular dystrophy

characterized by rapid progression of muscle degeneration which occurs early in life.

‘scoliosis’

Duchenne’s muscular Duchenne’s muscular dystrophydystrophy

Mutations in DMD gene lead to non functional dystrophin protein (localised on periphery of normal muscle fibres)

DMD patient

Normal

Topology of DNA

DNA supercoiling: coiling of a coil

Important feature in all chromosomes

Supercoiled DNA moves faster than relaxed DNA

Allows packing / unpacking of DNA

negatively supercoiled

Results from under or unwindingImportant in DNA packing/unpacking e.g during replication/transcription

positively supercoiled

Results from overwinding

Also packs DNA but difficult to unwind

Supercoiling topologySupercoiling topology

No supercoiling (left) to tightly supercoiled (right)

Visualising DNA/RNA with dyes

Ethidium bromide

EBr

supercoiled

Relaxed circle

Full length linear

Supercoiling explains why an uncut plasmid gives more than one band on a gel

DNA supercoiling takes 2 forms DNA supercoiling takes 2 forms toroidal (DNA around histones) or toroidal (DNA around histones) or

interwound (bacterial chromosomes)interwound (bacterial chromosomes)

Forces stabilising nucleic acid structures

Applications in polymerase chain reaction (PCR)

A) Denaturation and renaturation of DNA

The forces that stabilise nucleic acids (N.As) are largely common to those that stabilise proteins

The way they combine gives N.As very different properties

Denaturation of DNA

Also called melting

Occurs abruptly at certain temperatures

Tm – temp at which half the helical structure is lost

DNA melting curve

Tm varies according to the GC content

High GC content - high Tm

GC rich regions tend to be gene rich

Renaturation of DNA

Also called annealing

Occurs ~ 25oC below Tm

Property used in PCR and hybridisation techniques

Forces stabilising nucleic acid structures

B) Sugar-phosphate chain conformations

Fig: 28-18: Voet and Voet

Endo conformation (same side as C5’)B-DNA is C2’ endo

Conformation determined by 7 angles ( )

The out of plane atom is usually C2’ or C3’

1. N-glycosidic linkage has only one or two stable positions (syn/anti)

2. Sugar ring puckers to relieve crowding of substituents that would otherwise occur in planar conformation

Planar

Puckered

Forces stabilising nucleic acid structures

C) Base pairing

When monomeric A and T are co-crystallised:- They form Hoogsteen geometry

(C) Base pairing

D. Factors that stabilise N.As (c)

• Watson-Crick geometry is preferred in double helices due to various environmental influences

TA

Hoogsten base pairs stabilise tRNA tertiary structure

Forces stabilising nucleic acid structuresD) Base stacking and hydrophobic

interactions

Under aqueous conditions• Bases aggregate due

to the stacking of planar molecules

• This stacking is stabilised by hydrophobic forces

Forces stabilising nucleic acid structures

Tm of a DNA duplex increases with cationic concentration

Caused by electrostatic shielding of anionic phosphate groups

e.g. Mg 2+ more effective than Na+

E) Ionic interactions

Functions of nucleic acids

1) Storage of genetic information

2) Storage of chemical energy e.g. ATP

3) Form part of coenzymes

e.g. NAD+, NADP+, FAD and coenzyme A

4) Act as second messengers in signal transduction

e.g. cAMP

Functions of nucleic acids

1) Storage of genetic information

DNA is the hereditary molecule in almost all cellular life forms. It has 2 main functions:

Replication (making 2 copies of the genome) before every cell division

Transcription: process of copying a portion of DNA gene sequence into a single stranded messenger RNA (mRNA)

RNA (ribonucleic acid)

Has a more varied role. 4 main types of RNA are 1) mRNA: directs the ribosomal synthesis of

polypeptides and other types of RNA (translation)2) Ribosomal RNA: have structural & functional roles3) Transfer RNA: deliver amino acids during protein

synthesis 4) Ribonucleoproteins: take part in post

transcriptional processing

ATP (adenosine triphosphate)

Involved in1) Early stages of

nutrient breakdown

2) Physiological processes

3) Interconversion of nucleoside triphosphates

Functions of nucleic acids 2) Storage of chemical energy e.g. ATP

Functions of nucleic acids3) Form part of coenzymes

e.g. NAD+, NADP+, FAD and coenzyme A

Functions of nucleic acids4) Act as second messengers in signal transduction

e.g. cAMP (cyclic Adenosine Mono Phosphate)

Primary intracellular signalling molecule (second

messenger system)

Glycogen metabolism

cAMP dependent kinase (cAPK)

Gluconeogenesis

Fatty acid metabolism - thermogenesis