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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