1

• DNA that contains functional information for the synthesis of RNA or Protein → gene

• Ribosomal RNA’s (rRNA’s)→ complex that synthesizes proteins

• Messenger RNAs (mRNA’s) → intermediaries → carrier of genetic Info. from 1 or may genes to the ribosome

• Transfer RNA’s (t-RNAs) → translate mRNA to protein sequence

• Nucleotides and Nucleic AcidsNucleotides and Nucleic Acids

2

NucleoNucleotidestides have 3 features: have 3 features:

• Nitrogen containing bases• Pentose (5 C- sugar)• Phosphate moietyPhosphate moiety

Molecule WITHOUTPO4 → NucleoNucleosideside

NB: ‘ (primers) give to distinguish from C in purine/pyrimidines

EsterEster bondbond

3

• Purine and Pyrimidine BasesPurine and Pyrimidine Bases

N--glycosyl bond with the 1’ C in the pentose (previous slide)

Break double bond

Proton extraction

Pentose + Purine/Pyrimidine O-glycosidic bond (Nucleoside) + phosphate Nucleotide

4

• PurinesPurines

• Present in BOTHBOTH DNA and RNA

(A)(A) (G)(G)

5

• PyrimidinesPyrimidines

• Cytosine (C) found in BOTHBOTH DNA and RNA• Thymine (T) found ONLYONLY in DNA• Uracil (U) found ONLYONLY in RNA Distinguishing features

A, T G and C nucleotides → genetic information

6

• Two kinds of Pentoses present……Two kinds of Pentoses present……

H

Deoxy → deoxyribo(nucleotides/sides)

→ ribo(nucleotides/sides)

7

8

9

• Phosphodiester Bonds link Successive Nucleotides in Nucleic acidsPhosphodiester Bonds link Successive Nucleotides in Nucleic acids

• DNA and RNA nucleotides linked via PO4- grp 5’→ 3’-OH grp of the next nucleotide (phosphodiester linkage)

• Backbone of alternative linkages b/w PO4 and pentose grps

• Backbone is hydrophilic → -OH in CHO/carbohydrate undertake H-H bonding with H2O

• PO4 gp pKa = 0, pH 7 → -ve charged → interactions with +ve charges (proteins/metals/polyamines) [Expt. - Adding salt interfers with these interactions = ability to separate DNA from other cellular constituents]

• ALL phosphodiester linkage have the SAME orientation 5’ → 3’ (polarity)

• 5’ end → LACKS a nucleotide at position 5’

• 3’ end → LACKS a nucleotide at position 3’

10

DNA/RNA

Slow/non-enzymatichydrolysis

Breaking phosphodiester bonds

• RNA is more unstable → rapid breakdown in base(via the 2’-OH)

• DNA does NOTNOT(2’-OH gp is absent)

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RNA Breakdown

12

pA-C-G-T-AOH

pApCpGpTpApACGTA

Written in the 5’→ 3’ direction (left to right)

Oligonucleotides (<50 nucleotides; >50 – polynucleotides)

Nomenclature of nucleotides – what’s in a name…

Maybe written as:

MBBE 402 Lecture 15 13

14

• DNA is “defined”

• DNA base composition varies at a inter-special level• Same species, different tissues have the same DNA base composition• DNA base composition does not change• ALL DNA follows these simple rules:

A = T G = C

Sum of purines = Sum of pyrimidines: A + G = T + C

(Chargeff’s rules)

The famous double helix – solved by x-ray diffraction; insight that molecule was helical; 2 periodicities 3.4Å and 34Å

Solve the structure?

A = TG = C

• DNA StructureDNA Structure

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• Watson and Crick – the master the minds of the jigsaw puzzleWatson and Crick – the master the minds of the jigsaw puzzle

2 helical DNA chain wound around an axis, forming a right handed double helix

Hydrophobic backbone of alternative deoxyribose and PO4 gr on outside

Pyrimidines/purines stacked inside the double helix (hydrophobic and planar ring structure perpendicular to long axis

Offset pairing of 2 stands causes major and minor groove

Each paired nucleotide shares the same plane

3 H-H bonds b/w G/C ( G≡C) ;2 b/w A/T (A=T)DNA with high G≡C content difficult

to denature10 base pairs/turn* *crystal form

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Complementary strands; two anti-parallel strands containing not identical base bases, yet base pair which fitthe A=T and G≡C rules of engagement.

Replication

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• The Chemical Properties of DNAThe Chemical Properties of DNAHighly viscous at Room Temp., pH 7.0pH or Temp. (>80oC) → in viscosity

Reflect in structural changes DenaturationMelting→ disruption of H-H bonding b/w base pairs

DS → SS (no covalent bonds are broken)

Renaturation is a rapid one step process in undone DS DNA

Temp/pH return to physiological conditions→ annealing → intact DS

In SS DNA, two step process which is slower: “finding” then “zippering”

DS – double stranded

SS – single stranded

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• Free nucleotides have the highest UV Absorbance (Abs.)

• Once in sequence, stacking of the base pairs ↓ the Abs. (same number/types of nucleotides as above)

• Abs. is ↓↓ with complementary pair formation

• Denaturation of DS ↑ Abs. HYPERCHROMIC EFFECT

• Thus transition from DS to SS can be monitored by UV Abs.

Some areas more susceptible to denaturation that others…

Here we can measure the increase and decrease in DNA DS and SS via UV light

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• DNA (viral/Bacterial) denature when heated slowly

• Each DNA species has a specific denaturation temp. (Tm) which is reflective of the base pair composition

• Higher G≡C content; higher Tm (3 H-H bonds)

• A=T bonds require less H to denature

• Tm determination can provide estimation of base-pair content

• Region with high T=A, denature first (“bubbles”)

20

• Nucleic Acids from different species can form HybridsNucleic Acids from different species can form Hybrids

The process of complementary sequences in DNA strands can be used to detect similar DNA via duplex formation

Denaturing and renealling, complementary regions will bind/seek each other..

Small % will form a mixture of two strands from species – hybrid duplexes→ common evolutionaryHeritage, conserved proteins (i.e. conserved DNA/RNA)

Gene detection – hybridize ‘seeking’ labeled nucleotide sequence which are site specific.

CSI – DNA matching (bone fragments etc.)See changes in UV abs. Thus know if you have a ‘match’

Only small No. will form hybrids

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• UV light/radiation…..(200-400nm)UV light/radiation…..(200-400nm)

2 adjacent Ts

Breaking double bond

Dimer

Similar effect:• Radiation (C14, H3, X-rays etc.)• Chemicals/alkylating agents

Carcinogens

22

What does it mean?

23

• Sequencing DNA via Sanger MethodSequencing DNA via Sanger Method

Separating and identifying DNA strands that differ by a single nucleotide via gel electrophoresis

Primer stand has single “non-native” Nucleotides added to it

24

H Stops DNA synthesis → a freeze frame of the process of DNA\sequence chain elongation

P

OH

BASE

(radio labeled nucleotide analogue)

Add these to our growing sequence

(Dideoxynucleoside)

MBBE 402 Lecture 15 25

Analyze the primer at different stages of 5’ chain elongation

Differentmasses

separate

LabeledNucleotides

Freeze frame

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• Nucleotides carry chemical energy in cellsNucleotides carry chemical energy in cells

Mono-PO4

Di-PO4

Tri-PO4

Sequential hydrolysis

Energy

27

• ATP is the most common source for driving energy requiring cellular RATP is the most common source for driving energy requiring cellular Rxx

Phosphoanhydride bonds = 30 KJ/mol

= 14 KJ/mol

28

• Adenine Nucleotide are components for many Enzymes cofactorsAdenine Nucleotide are components for many Enzymes cofactors

Removal of Adenine → loss of co-factor activity

(Vitamin B2)

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• cAMP, cGMP and ppGpp – used in extracellular chemical signalingcAMP, cGMP and ppGpp – used in extracellular chemical signaling

Secondary messengers – target specific membrane bond receptors, which active a cascade of internal cellular events via G-coupled proteins.

(bacteria)