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DNA and RNAStructure and Function

DNA and RNAStructure and Function

RNA and DNA Chemical Structures

DNA Structural Elements

RNA Structural Elements

Cleavage of DNA and RNA by Nucleases

Nucleic Acid-Protein Complexes

RNA and DNA Chemical Structures

DNA Structural Elements

RNA Structural Elements

Cleavage of DNA and RNA by Nucleases

Nucleic Acid-Protein Complexes

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The nucleic acidsThe nucleic acids

Nucleic acids are complex structures used to maintain genetic information.

DNADNA deoxyribonucleic acidServes as the “Master Copy” for

most information in the cell.

RNARNA ribonucleic acidSeveral types. Overall, it acts to transfer information from DNA to the rest of the cell.

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DNA and RNA compositionDNA and RNA composition

Primary structure of both materials is very similar.

Each consists of a sugar/phosphate backbone with nitrogenous bases attached.

Major difference is in the type of sugar and bases used.

sugar phosphate

base

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Sugars usedSugars used

OHOCH2

H HHH

OH OHOH

OHOHOCH2

H HHH

OH HH

OH

riboseused in RNA

deoxyriboseused in DNA

Not a verybig difference!

Not a verybig difference!

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NucleosideNucleoside

A sugar - base combination.

OHOCH2

H HHH

OH H

BaseBase

SugarIn this case deoxyribose

SugarIn this case deoxyribose

-N-glycosidiclinkage

-N-glycosidiclinkage

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The nitrogenous basesThe nitrogenous bases

Five bases are used that fall in two classes

PurinesPurinesA double ring (6 and 5 membered) structure

Includes adenine and guanineUsed by both DNA and RNA

PyrimidinesPyrimidinesA six membered ring structure

Cytosine is used in both DNA and RNA

Thymine is used in DNA, Uracil used in RNA

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The nitrogenous basesThe nitrogenous bases

NC

C

CCN

N

NH

CH

NH2

|

H

NC

C

CCN

N

NH

CH

O||

H

H2N

NC

C

CCNH

O

NH2

|

H

H NC

C

CCNH

O

H

H

O|| CH3

NC

C

CCNH

O

O||

HH

H

adenineadenine guanineguanine

cytosinecytosine thyminethymine uraciluracil

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NucleotidesNucleotides

Produced if the -OH on the sugar of a nucleoside is converted into a phosphate ester.

NC

C

CCN

N

N

CH

NH2

|

H

O-O-P-O-CH2

H HHH

OH H

| O-

O||

deoxyadenosine monophosphate(dAMP)

deoxyadenosine monophosphate(dAMP)

Each is named based on sugar and base nameand then the number ofphosphates is indicated.

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Primary structurePrimary structure

Phosphate bonds link DNA or RNA nucleotides together in a linear sequence.

3’,5’-phosphodiester

O|

O -- P -- O --||O

-CH2 O

OH

O|

O -- P -- O --||O

-CH2 O

O|

O -- P -- O --||O

-CH2 O

O|

O -- P -- O --||O

-CH2 O

NC

C

CCN

N

N

CH

NH2

|

H

NC

C

CCNO

NH2

|

H

H

NC

C

CCNO

H

H

O|| CH3

NC

C

CCN

N

N

CH

O||

H

H2N

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Primary structurePrimary structure

It is cumbersome to draw complete nucleic acids so a simplified set of lines can be used.

A G C T (U)base

sugar1’

5’

nucleosides

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Primary structurePrimary structure

Representation of an oligonucleotide showing the 3’,5’-phosphodiester bonds.

A G C T (U)

3’ end5’ end

PPPHO OH

A very abbreviated representation is to simply listthe base sequence starting from the 5’ end - AGCT

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DNADNA

In native DNA, a large number of nucleotides are linked together.

The size and conformation of the molecule is species dependent.

In simple prokayrotic cells, a single strand is produced.

In more complex species, multiple strands are used. Each is combined with proteins called histones.

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DNADNA

Number of LengthOrganism Base Pairs (m) Conformation

VirusesSV40 5,100 1.7 circularAdenovirus 36,000 12 linear phage 48,600 17 circular

BacteriaE. coli 4,700,000 1,400 circular

EukaryotesYeast 13,500,000 4,600 linearFruit fly 165,000,000 56,000 linearHuman 3,000,000,000 1-2 x 106 linear

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RNARNA

Approximately 5-10% of the total weight of a cell is RNA. DNA is only about 1%

RNA exists in three major forms.

• Ribosomal RNA - rRNA.Ribosomal RNA - rRNA. Combined with protein to form ribosomes, the site of protein synthesis.

• Messenger RNA - mRNA.Messenger RNA - mRNA. Carries instructions from a single gene from DNA to the ribosome.

• Transfer RNA - tRNA.Transfer RNA - tRNA. Forms esters with specific amino acids for use in protein synthesis.

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DNA structural elementsDNA structural elements

Sugar-phosphate backboneSugar-phosphate backboneCauses DNA chain to coil around the outside of the attached bases like a spiral staircase.

Base PairingBase PairingHydrogen bonding occurs between purines and pyrimidines. This causes two DNA strands to bond together.

adenine - thymine guanine - cytosineadenine - thymine guanine - cytosine

Always pair together!Always pair together!

Results in a double helix structure.

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Base pairing and hydrogen bondingBase pairing and

hydrogen bonding

N

N

O| |

- H

N - H

N

NN

N

O| |

H - N

N

N O| |

O| |

H3C

- H

guaninecytosine

thymine adenineN

N N

N|

HH

N

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Hydrogen bondingHydrogen bonding

Each base wants toform either two or three hydrogen bonds.

That’s why only certain bases will form pairs.

G

T

C

A

C G

A

C

T

G

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The double helixThe double helix

The combination of the stairstep sugar-phosphate backbone and the bonding between pairs resultsin a double helix.

The combination of the stairstep sugar-phosphate backbone and the bonding between pairs resultsin a double helix.

Distance betweenbases = 0.34 nm

10.5 bases/turn

2 nmbetweenstrands

One complete

turn is 3.4 nm

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Double helixDouble helix

The original Watson and Crick model for the double helix, B-DNA, is one of several conformations.

B-DNAB-DNA - believed to be the predominate form under physiological conditions. It has 10.5 bases per turn.

A-DNAA-DNA - formed when B-DNA is chemically treated. It has 11 bases per turn.

Z-DNAZ-DNA - left handed helix with 12 bases per turn. It may play a role in regulation of gene expression.

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Physical and biologicalproperties of the double helix

Physical and biologicalproperties of the double helix

Inspection of the DNA double helix indicates a mechanism for its replication and transfer of information.

3’

3’

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Physical and biologicalproperties of the double helix

Physical and biologicalproperties of the double helix

It is possible to overcome the hydrogen bonding and van der Waal forces that hold the strands together - denaturingdenaturing.

heat, acids, bases or organic solventsheat, acids, bases or organic solvents

The strands tend to reassociate if the source of denaturing is removed.

ExampleExample - heating DNA will cause it to unwind. It will recoil when cooled - annealing.

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Tertiary structure of DNATertiary structure of DNA

Studies of native, intact DNA have revealed two distinct forms - linear and circular.

The circular form is the result of covalent joining of the two ends of a linear double helix.

It can exist in two conformations

RelaxedRelaxed

SupercoiledSupercoiled

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Tertiary structure of DNATertiary structure of DNA

Relaxed Supercoiled

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Supercoiled DNASupercoiled DNA

• The supercoil is not simply a coil of the circular form, there is extra twisting.

• While it is less stable than the relaxed form, there is evidence to show that it exists in vivo.

• TopoisomerasesTopoisomerases - enzymes that catalyze changes in the topology of DNA have been isolated.

• This form may play a regulatory role in DNA replication or represent a more compact form for storage.

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RNA structural elementsRNA structural elements

Two fundamental differences between the primary, covalent structures of RNA and DNA.

• RNA contains ribose rather than 2-deoxyribose.• RNA uses uracil instead of thymine.

The extra hydroxyl group in RNA makes it The extra hydroxyl group in RNA makes it more susceptible to hydrolysis.more susceptible to hydrolysis.

This may be the main reason that DNA is the ultimate repository of genetic information.

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RNA structural elementsRNA structural elements

• RNA always exists as single-stranded molecules.

• It does not take on an extended, secondary structure like DNA.

• The strands tend to fold into a uniform, periodic pattern.

• Several structural elements are observed.

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RNA structural elementsRNA structural elements

Hairpin turnsHairpin turnsLoops in the chain that bring together complementary base pairs. If long enough, a double helix region is observed.

Right-handed double helixRight-handed double helixResult from intrastrand folding.

Internal loops and bulgesInternal loops and bulgesRelatively common in RNA molecules. These are structural features that disrupt the formation of continuous double helix regions.

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RNA structural elementsRNA structural elements

Bulge Internalloop

Helicalsegments

Unpairedloop at tip

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tRNA structuretRNA structure

The smallest typeof RNA. It consistsof 74-93 nucleotides.

These moleculesare the carriers ofthe 20 amino acidswith at least one tRNAfor each.

They often containseveral unusualpurines or pyrimidinebases -modificationsof the basic four.

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tRNA structuretRNA structure

All tRNA have a common 2o and 3o structure.

A

C

C

A

C

C

U

C

G

U

CU

U

C

G

G

G

G

G

CC GGG

CC GG

A CGG

CC GGU

C

C

C

C

U

C

A

U

G

G

A

G

G

G

G

GU

U

CC G

U

C GC

AU

G

G

C

U

AG U

A GU

G

GC

H O- 3’

5’

anticodon

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rRNArRNA

These molecules have 2o and 3o structure similar to tRNA. However, they are much larger.

16S rRNAE. Coli

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Cleavage of DNAand RNA by nucleases

Cleavage of DNAand RNA by nucleases

Characterization of nucleic acid structure and function often requires breaking these large molecules into fragments.

• Primary structural analysis of DNA/RNA

• Site specific cleavage for recombinant DNA manipulation.

Historically, hydrolysis by acids (for DNA), bases (for RNA) and enzymes has been used.

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Cleavage of DNAand RNA by nucleases

Cleavage of DNAand RNA by nucleases

NucleasesNucleases• Enzymes that catalyze the hydrolysis of

phosphodiester bonds. • Normally used to catalyze degradation

of damaged or aged nucleic acids.• Some work on both DNA and RNA and

others are substrate specific.

DNasesDNases - deoxyribonucleases, act on DNA.

RNasesRNases - ribonucleases, act on RNA.

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Cleavage of DNAand RNA by nucleases

Cleavage of DNAand RNA by nucleases

NucleasesNucleases• ExonucleasesExonucleases - catalyze the removal of

terminal nucleotides, 3’ and 5’ types.

• EndonucleasesEndonucleases - catalyze removal of internal phosphodiester bonds.

Type aType a - act on the 3’ hydroxyl group of a nucleotide with the phosphorous group.

Type bType b - act on the 5’ hydroxyl group of a nucleotide with the phosphorous group.

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Cleavage of DNAand RNA by nucleases

Cleavage of DNAand RNA by nucleases

EndonucleasesEndonucleases

A G C T

3’ end5’ end

PPPHO OH

Type a

Type b

snake venomphosphodiesterase

While these enzymes are useful for cutting DNA andRNA into manageable sizes, they are not very specific.

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Properties of nucleasesProperties of nucleases

Enzyme Substrate Type Specificity

Rattlesnake venom DNA,RNA exo(a) 3’ end, no basephosphodiesterase specificity.

Spleen DNA,RNA exo(b) 5’ end, no basephosphodiesterase specificity.

Pancreatic RNA endo(b) 3’ side preference ribonuclease A for pyrimidines.

Spleen DNA endo(b) Internal esterdeoxyribonuclease II bonds. No base

specificity

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DNA restriction enzymes DNA restriction enzymes

Restriction endonucleasesRestriction endonucleases• Restriction enzymes.• Most specific enzymes for DNA cleavage.• Recognize specific base sequences.

These enzymes are produced in bacterial cells and act to degrade or “restrict” foreign DNA molecules.

Host DNA is protected because some bases near the cleavage sites are methylated.

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DNA restriction enzymes DNA restriction enzymes

Several hundred restriction enzymes have been isolated and characterized.

Nomenclature.Nomenclature.• Three letter abbreviation representing the

source species.

• A letter representing the strain.

• A roman number for the order of discovery.

EcoEcoRIRI - The first restriction enzyme isolated from E. coli, strain R.

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DNA restriction enzymes DNA restriction enzymes

Enzyme Sequence

EcoRI 5’ - GAATTC

BaII 5’ - TGGCCA

TaqI 5’ - TCGA

HinfI 5’ - GANTC

Enzyme Sequence

EcoRI 5’ - GAATTC

BaII 5’ - TGGCCA

TaqI 5’ - TCGA

HinfI 5’ - GANTC

These enzymes areall DNA specific ofthe type endo (a).

The red line indicatesthe point of cleavagefor the recognitionsequence.

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DNA restriction enzymesDNA restriction enzymes

Catalysis of DNA into smaller fragments can be used for:

• Making it easier to assay DNA

• IdentificationResults in a unique set of fragments

for different DNA molecule.Each individual will produce a unique set of fragments - DNA fingerprints.

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Nucleic acid - protein complexesNucleic acid - protein complexes

These are examples of ‘supramolecular assemblies’ - organized clusters of macromolecules.

Significant examples are nucleoproteins- complexes of protein and nucleic acids.

ExamplesExamples VirusesViruses

ChromosomesChromosomes

snRNPssnRNPs

RibosomesRibosomes

Ribonucleoprotein enzymesRibonucleoprotein enzymes

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VirusesViruses

Stable, infective particles composed of a nucleic acid (DNA or RNA) and protein.

The protein subunits form a protective shell around the nucleic acid

core.

Larger, complex viruses also have an additional envelope of glycoproteins and membrane lipids.

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VirusesVirusesDiagram of the human immunodeficiency virus (HIV).

Glyco-proteins

Proteinsubunits

Reversetranscriptase

RNA

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ChromosomesChromosomes

Eukaryotic chromosomes consists of DNAstrands coiled around protein - histones.

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ChromosomesChromosomes

The normal number of chromosome pairs varies among the species.

AnimalAnimal Pairs Pairs PlantPlant PairsPairsMan 23 Onion 8Cat 30 Rice 14Mouse 20 Rye 7Rabbit 22 Tomato 12Honeybee, White pine 12

male 8 Adder’s 1262female 16 tongue fern

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ChromosomesChromosomes

• DNA is tightly wrapped around histones which are relatively small proteins.

• The resulting complex is called chromatin.

• Eight histones are used to form the basic core for the DNA to wrap around.

• The spooled complexes are called nucleosomes with about one million per human chromosome.

• The nucleosomes continue to wind, forming chromatids.

• The chromatids combine resulting in a chromosome unit.

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HistoneHistone

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Coiling of DNACoiling of DNA

DNA

‘Beads-on-a-string’ formof chromatin

30 nm Fiber

One coil30 rosettes

One loop5 x 107 bp

Two chromatidsare produced from2 x 10 coils.

They are not shownin this figure.

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snRNPssnRNPs

• A new class of ribonucleoprotein complexes

• Active clusters of small nuclear ribonucleic acids (snRNAs) and protein.

• They are only found in eukaryotic cells.

• They catalyze specific splicing that removes exon sequences to produce mature mRNA.

• Analogous cytoplasmic particles are scRNPs.

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snRNPssnRNPs

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Other complexesOther complexes

RibosomesRibosomesServe as the platform for protein synthesis. They consist of about 65% RNA and 35% protein. There are two major subunits that dissociate during the translation process.

Ribonucleoprotein enzymesRibonucleoprotein enzymesAn important class of enzyme. It includes ribonuclease P and telomerase.

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Ribonuclease ARibonuclease A