Molecular Biology

Biochemistry Cell BiologyGenetics

Nucleic Acid Structure

• Objectives:

To know and identify the structure, roles and classifications of

• bases• sugars• nucleosides• nucleotides• DNAs• RNAs

Bases

Nucleic Acids / Bases/ Definition

Nucleobases are:

Aromatic (planner)

Heterocyclic, Nitrogen containing

Classified into:

Purine

Pyrimidine(planner character facilitates their close (planner character facilitates their close

association or stacking which stabilizes association or stacking which stabilizes

double stranded DNA)double stranded DNA)

Hetero atoms are Hetero atoms are referred to referred to atoms other atoms other than C and H. than C and H. In biology they In biology they are N, O and Sare N, O and S

Nucleic Acids / Bases/Structure

Pyrimidine

Imidazole

By the attachment of differentBy the attachment of different

groups to the rings, different typesgroups to the rings, different types

of Py and Pu are generated.of Py and Pu are generated.

NA / Bases/ Classification

• Purine Bases (pu): Purine Bases (pu): • Major : (A)&(G)Major : (A)&(G)• Minor: Inosine(I) & Minor: Inosine(I) & methyl guanine(7mG) methyl guanine(7mG) • Unnatural : Unnatural :

MercaptopurineMercaptopurine,,

Allopurinol & Allopurinol &

8-Azaguanine 8-Azaguanine

• Pyrimidines (py): Pyrimidines (py):

• Major : (T), (C) & (U) Major : (T), (C) & (U)

•Minor: DHU , 5mC & 5hmCMinor: DHU , 5mC & 5hmC

•Unnatural: Fluorouracil (5FU) & Unnatural: Fluorouracil (5FU) & 6-aza cytosine( AZC) 6-aza cytosine( AZC)

Hot spot

NA / Bases/ Classification/ Py

NA / Bases/ Isomerism/ Tautomerism

Diffinition:Diffinition: is a kind of is a kind of isomerism between isomerism between amine-imine or keto amine-imine or keto (oxo)- enol groups.(oxo)- enol groups.

Types:Types: Lactam( Keto, Lactam( Keto, Amine) Lactyme (Enol, Amine) Lactyme (Enol, Imine)Imine)

Properties:Properties: They have They have different hydrogen different hydrogen bonding patternsbonding patterns

Physiologic Types:Physiologic Types: amino and oxo forms amino and oxo forms

Tautomerism/ importance in mutagenesis

Tautomerism

Lactam Lactyme

Keto Imine Enol Amine

• In physiological Condition: A/T and G/C• Rare tautomers of G may pair with A, U with C, A

with U or G and C with U

Sugars

Nucleic Acids / Sugars

D-family

Aldo pentose

Furanose

β-Anomer

Ribose or deoxy Ribose

Numbered by Prime

Isomerism ( 2’ endo in DNA , 3’ endo in RNA)

Nucleosides

Nucleic Acids / Nucleosides

Structure:

Isomerism:

Nomenclature:

Roles :

Classification:

• β-N-glycosidic linkage of a base and a

pentose (1‘ to 9 in PU and 1‘ to

1 in PY).

• syn or anti conformation

• Name of the base + suffix sine for Pus(adenosine & guanosine)

• dine for PYs (uridine & thymidine)

• They are major part of nucleosides.

• Synthetic forms are used as drugs (Cytarabin)

Major:: Cytidine & deoxy cytidine.

• There are 8 major nucleosides.

Minor : ribothymidine (rT) & psudouracil (ΨU).

Unnatural : Cytarabin.

Nucleotides

Building block

Coenzymes (NAD, FAD & Co-A)

Group transfer

Energy carrier ( ATP & GTP)

Regulatory roles: Drugs

• in lipid synthesis( CDP-acylglycerol)in lipid synthesis( CDP-acylglycerol)• in sugar synthesis( UDP-glucose)in sugar synthesis( UDP-glucose)• in protein synthesis(tRNA)in protein synthesis(tRNA)• methyl donor as SAM methyl donor as SAM

(S-adenosylmethionine)(S-adenosylmethionine)

NA/ Nucleosides/ Biomedical importance

•Second messenger for hormones(cAMP or GMP)•Allosteric regulator of many enzymes(ATP•& AMP in metabolic pathways)

• Synthetic pu & py analogs that contain Halogens, thiols or additional nitrogen are employed in:

• treatment of gout (Allopurinol)• Chemotherapy of cancer ( cytarabin)• AIDS treatment ( azathioprine)• Immunosuppression responses

during organ transplantation ( azathioprine)

Nucleic Acids / Nucleotides

Structure:

Isomerism:

Nomenclature:

Roles:

Classification:

• Nucleoside+ Phosphate group(s

=phospho ester of nucleosides• In most cases the phosphate

group is linked to the 5’ carbon.• They may have 1, 2,or 3

phosphate groups.

They may have syn or anti conformation They may have syn or anti conformation

with with predominate anti conformerpredominate anti conformer..

1-Name of the nucleoside1-Name of the nucleoside

+ the number of phosphate group.+ the number of phosphate group.

2-Name of their corresponding acid2-Name of their corresponding acid

such as thymidylic or guanylic acidsuch as thymidylic or guanylic acid..

• Building block• Coenzymes (NAD, FAD & Co-A)• Group transfer• Energy carrier ( ATP & GTP)• Regulatory roles: • Drugs

MajorMajor:: CMP, dCMP, CDP, CTPCMP, dCMP, CDP, CTP

There are 24 major nucleotidesThere are 24 major nucleotides

MinorMinor : : cAMP, cGMPcAMP, cGMP

UnnaturalUnnatural : : Cytarabin Cytarabin

Nucleic Acid:1-DNA2-RNA

Nucleic Acids /DNA/ Structure• Objectives To know and identify different levels of

DNA organization

1- Primary structure

2-Secondary structure

Different geometry of

base pairs & base steps

3-Higher order of DNA structure

in prokaryotes

in Eukaryotes

DNA / General facts• Its structure was discovered in 1953 by Watson

and Crick• It is a polyester compound• Has acidic character

It is a polymer in which the monomers (nucleotides)are joined by PDE bonds between5’ and 3’ carbon atoms of two successive nucleotides.

Because of the phosphate

moiety, they have acidic

character (negatively charged).

• Has polarity

• It is double helix

DNA / General facts

DNA has end-to-end

chemical Orientation

(3’ and 5’ ends) and

by convention it

is written in the

5’ 3’ direction

• The two strand are:• 1- in opposite polarities• 2- stabled by different

bonds:

Hydrophobic bond

H bond

Different bonds and interactions in

• Covalent:

PDE bonds in the backbone

• Hydrogen: between complementary bases Primary or Natural (Watson-Crick) Secondary or Hoogsteen pairing (non-Watson-Crick)

• Hyrophobic (van der Waals)

between the stacked adjacent base pairs.

DNA / General facts

Particular region bound to protein clearly depart from the standard conformation

• Has specific groove (s)• It is flexible about its long axis it

• It may be linear or circular

DNA / Different ways to show primary structure

5’ 3’

Primary structure is a huge linear polymer of dNTPs that are joined to each other by 5’-3’ PDE bonds.

Secondary structure of DNA / Different ways to show it

5’ 3’

3’ 5’

Secondary structure is formed by base pairing between two complementary strands. It may be B, A or Z form.

What about DNA grooves

• They are generated because of the angle of base pairs

• They are important for the interaction with proteins

DNA denaturation ( melting)

DNA Renaturation DNA Renaturation (annealing)(annealing)

&&

DNA denaturation ( melting)

• Definition: Strands unwinding and separation.

In linear DNA both strands unwind and separate.

In circular DNA the two strands can not unwind and separate, they make

an interlocked, tangled mass of singlestranded DNA. When there is

a nick the strands will separate by melting

DNA renaturation (annealing)

• Definition: The reassociation of the

two separated strands by slow and gentle removing of Denaturating agents.

It may lead to:• Homo duplex• Randon coil• Hetero duplex

Denaturating agents/ TDenaturating agents/ Tmm

Definition: Tm is referred to the temperature

that 50% of dsDNA is denatured.

Tm depends on: 1- G C percent 2-Solutions of low ion

concentration 3- Alkaline solution 4-Formamide or urea

(concentrated solution)

1. DNA molecule is very long and has to be contained within cells that are so small.

2. In free form the repulsion of negatively charged DNA is responsible for the large volume occupied by DNA.

3. Chromosomes are compacted DNAs.

4. Double helices can be compacted and form higher order structures that are referred to super helices which are shorter and thicker.

5. In cells there are some positively charged proteins that responsible for the compaction ( supercoiling) of DNA:

Histons in eukaryotes Histon like proteins in prokaryotes

Supercoiling/ general facts

Higher order of DNA structure Supercoiling/ Topoisomerism

• Definition:• Where it can be find:

• When it can be formed:• Different names: • Different types: The special type of isomerism

that is find in supercoiled DNA

Where the two ends of a DNA molecule are fixed , the molecule

exhibit a superstructure under certain conditions .When the base pairing is interrupted

and a local region unwind such as during replication, transcription and

binding of many binding protein to DNA.

superhelix supertwistsupercoil.

Relax: There is no superhelix turnPositive: The handness of double and

super helix are the sameNegative: The handness of double and

super helix are opposite

Supercoiling degreeSupercoiling degree

Prokaryotic DNAProkaryotic DNA• Is circular DNA• 1mm length• Is about 1000 fold compacted and form

a structure called nucleoide.

How It is compacted: 1-Supercoiling 2-Numerous small protein molecules

(H-NS) compact DNA, increase its sedimentation rate and decrease its viscosity.

Comparison of different DNA types

Chromatin

Euchromatin

Heterochromatin

[Transcriptionally active]

[Transcriptionally inactive]

Operational Classification

Structural genes, rRNA genes, regulatory regions, etc.

Centromeric chromatin Attachment sites to nuclear matrix

Human beings have roughly 3 billion base pairs of DNA in 23 chromosomes (haploid)

Yeast has 13 million base pairs in 17 chromosomes

Distance between bases is 3.4 Angstrom

For human this would be 3.4×3×109 Angstroms. This equals 1.02 meters per haploid genome, 2.04 meters per cell

The size of the nucleus is roughly 10 micrometers in diameter

DNA Compaction in eukaryotes: General facts

Histones/ Definition & their Histones/ Definition & their conservationconservation

• They are small positively charged proteins that bind to the DNA

• They are highly conserved * The aa sequence of H2a, H2b, H3 and H4 are

remarkably similar among distantly related species. For example H3 of sea urchin and calf thymus are identical except for one aa.

* H1 is more variable. In certain tissues such as nucleated RBC of birds H1 is replaced by H5.

* The similarity between histones of all eukaryotes indicates its optimized function and same tridimentional structure of DNA in all of them.

• Histones are the most abundant proteins that are responsible for DNA compaction.

• The total mass of the histones associated with DNA in chromatin is about equal to that of DNA.

Histones/ AmountsHistones/ Amounts

Histones/Histones/ Classification & structure Classification & structure

• Classification:• H1• H2a• H2b• H3 • H4& • Numbers of their variants

PDB

Histon type MW Conservation aa number Lys+ Arg%Histon type MW Conservation aa number Lys+ Arg%

H1H122,50022,500Low 244Low 24430.830.8

H2AH2A13,96013,960Mod 129Mod 12920.220.2

H2BH2B13,77413,774Mod 125Mod 12522.422.4

H3H315,27315,273High 135High 13522.922.9

H4H411,23611,236High 02High 0224.524.5

Histones/ Classifications/ PropertiesHistones/ Classifications/ Properties

Histones/ PositioningHistones/ Positioning

• The sequence they prefer to attach

The distribution of histone genes in human genome

Their genes haveTheir genes have not any intron not any intron . .Their mRNA has Their mRNA has not any poly A tailnot any poly A tail..

Histone Structure and Nucleosome assemblyHistone Structure and Nucleosome assembly

Why Nucleosome? Because DNA and histones are very highly charged Because the nucleosome structure is very organized

How they assemble?

Mixing the components of the nucleosome will form a random aggregate rather than organized structure

The nucleosome assembly is mediated by many chaperon and protein modifying enzymes.

When they assemble?

Nucleosomes need to be assembled ROUTINELY during cell division following DNA replication

Nucleosomes ALSO need to be assembled ROUTINELY following inactivation of temporally expressed genes

Compaction of Eukaryotic DNA: first level (Nucleosome))

They cancel the negative charges of DNA, allowing it to be compacted.

They are a core for wrapping DNA around it.

They have regulatory role in gene expression

Histones/ RolesHistones/ Roles

Double helix

(Nucleosomes) 10 nm fibril

30 nm fiber

loops on scaffold

heterochromatin

chromosome

DNA Compaction in eukaryotes: General facts

Nucleosome with & without H1

+ +

++

++

- --

------ - - - - -

-

Types of Histone Modifications

• Acetylation:Convert +ve charge to neutral

• Methylation: Convert +ve charge to

neutral

• Phosphorylation

Convert +ve charge to

-ve

• Ubiquitinylation Convert +ve charge to

neutral

Histone Modification Enzymes

• Histone acetyl transferases (HATs) and Histone deacetylases (HDACs)

• Methylases and demethylases

• Kinases and phosphatases

• Ubiquitin conjugating enzyme (ubiquitin ligase) and deubiquitinating enzyme

The importance (Meaning) of Histone modification (Histone code)

RNA• Objectives:

To know and identify: Primary, secondary and Tertiary

structure of RNA. Ribozymes Structure and function of tRNA. Structure and function of rRNA. Structure and function of mRNA.

RNA Structure• It has ribose instead of deoxyribose so, it is

more labile and more reactive than DNA.• As a result of more liability, RNA is cleaved into

mononucleotides by alkaline solution.• It has uracil instead of thymine.• Like DNA it can be double/single stranded,

linear / circular.• Unlike DNA, RNA may have different

tridimentional structure.