Properties and functions of nucleic 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 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. 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

learning objectives Reference Compare sizes of DNA and understand the C-value paradox 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 Reference Chapter 29: Biochemistry (3e) by D Voet and J Voet (Wiley Publishing)

Outline 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

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

genome sizes organism Number of base pairs (kb) viruses   organism Number of base pairs (kb) viruses Lambda bacteriophage ( λ) 48.6 bacteria Eschericia coli 4,640 eukaryotes Yeast 13,500 Drosophila 165,000 Human 3.3 x 106  

What is the Human genome project? Goal: to sequence the entire human nuclear genome Public consortium Headed by F Collins Started in mid 80’s Working draft completed in 2001 Nature: Feb 2001 Celera Genomics Headed by C Venter Started in mid 90’s Working draft completed in 2001 Science: Feb 2001 Human genome = 3.3 X 10 9 base pairs Number of genes = 26 – 32,000 genes

Mitochondrial genome (16.6kbp) – multicopy, circular, ds DNA The 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-related 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? ....or in forensics what can a single human hair tell you? mitochondrial DNA Hair shaft nuclear DNA Hair root

Does size matter? C-value paradox Boa constrictor Genome size: 2.1 Gbp Homo sapiens sapiens Genome size: 3.2 Gbp mountain grasshopper Podisma pedestris Genome size: 18 Gbp protozoan Amoeba dubia Genome size: 670Gbp C value: DNA content of any haploid cell

Comparative genome sizes   C-value paradox   Why is there a discrepancy between genome size and genetic complexity?

Protein domains contribute to organism complexity Explaining the 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

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 dystrophy 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 Allows packing / unpacking of DNA Supercoiled DNA moves faster than relaxed DNA

Supercoiling topology No supercoiling (left) to tightly supercoiled (right) negatively supercoiled Results from under or unwinding Important in DNA packing/unpacking e.g during replication/transcription positively supercoiled Results from overwinding Also packs DNA but difficult to unwind

Visualising DNA/RNA with dyes EBr Ethidium bromide

Supercoiling explains why an uncut plasmid gives more than one band on a gel Full length linear Relaxed circle supercoiled

DNA supercoiling takes 2 forms toroidal (DNA around histones) or interwound (bacterial chromosomes)

Forces stabilising nucleic acid structures 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 A) Denaturation and renaturation of DNA Applications in polymerase chain reaction (PCR)

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

The out of plane atom is usually C2’ or C3’ 1. N-glycosidic linkage has only one or two stable positions (syn/anti) Conformation determined by 7 angles ( ) 2. Sugar ring puckers to relieve crowding of substituents that would otherwise occur in planar conformation Planar Puckered The out of plane atom is usually C2’ or C3’ Endo conformation (same side as C5’) B-DNA is C2’ endo Fig: 28-18: Voet and Voet

Forces stabilising nucleic acid structures C) Base pairing

Hoogsten base pairs stabilise tRNA tertiary structure D. Factors that stabilise N.As (c) (C) Base pairing When monomeric A and T are co-crystallised: - They form Hoogsteen geometry Hoogsten base pairs stabilise tRNA tertiary structure Watson-Crick geometry is preferred in double helices due to various environmental influences T A

Forces stabilising nucleic acid structures D) 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 mRNA: directs the ribosomal synthesis of polypeptides and other types of RNA (translation) Ribosomal RNA: have structural & functional roles Transfer RNA: deliver amino acids during protein synthesis Ribonucleoproteins: take part in post transcriptional processing

ATP (adenosine triphosphate) Functions of nucleic acids 2) Storage of chemical energy e.g. ATP ATP (adenosine triphosphate) Involved in 1) Early stages of nutrient breakdown 2) Physiological processes 3) Interconversion of nucleoside triphosphates

Functions of nucleic acids 3) Form part of coenzymes e.g. NAD+, NADP+, FAD and coenzyme A

Functions of nucleic acids 4) 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