Structure and Topology of DNA

Lecture 1 of Molecular Biology

Central Dogma

The flow of genetic information: DNA → RNA → Protein

(info storage → info conveyance → function)

Where is DNA found?

In nearly ever cell except for red blood cells.

Percent of genome coding for protein.

~1-2%

What evidence supported the DNA model?

X-ray diffraction data from Rosalind Franklin and Maurice Wilkins

Photo 51

X-shaped X-ray diffraction pattern indicating a helical structure.

Nucleotide components

Nitrogenous base, pentose sugar, and phosphate group.

Nucleoside vs Nucleotide

→ Nucleotide = Base + Sugar

→ Nucleotide = nucleoside + phosphate

Purines

Adenine (A), Guanine (G).

Pyrimidines

Cytosine (C), Thymine (T), Uracil (U).

DNA bases

A,T,G,C

RNA bases

A,U,G,C

DNA vs RNA

DNA:

• Double Stranded

• Deoxyribose

• H at 2’ carbon

• has thymine

• more stable

RNA:

• Single Stranded

• Ribose

• OH at 2’ carbon

• has Uracil

• Less stable

Cytosine deamination

C → U

Why is uracil in DNA a problem?

Uracil in DNA can damage the genetic code if not corrected.

Why DNA uses thymine instead of uracil?

Allows detection of cytosine deamination.

Pentose Sugars

• Ribose (RNA)

• Deoxyribose (DNA)

Sugar pucker

• DNA: C-2’ endo pucker

• RNA: C-3’ endo pucker

Why is the sugar pucker important?

It influences the A or B form helices. RNA generally adopts the compact geometry of A-form helices because of its C-3’ endo sugar pucker.

Phosphodiester bond

Covalent bond linking nucleotides.

Bonds formed between

• 3’-OH of one nucleotide

• 5’-phosphate of the next

Directionality of DNA/RNA

5’ → 3’, if direction is not given on the exam assume this.

Why RNA is less stable than DNA.

2’-OH participates in hydrolysis.

RNA hydrolysis

Occurs rapidly under alkaline conditions (high ph).

Watson-Crick base pairing.

• A = T (or U)

• G ≡ C

Hydrogen bonds of base pairs.

• A-T: 2 H bonds

• G-C: 3 H bonds

Base stacking

Hydrophobic interactions between planar bases.

Importance:

• Major stabilizing force of DNA

• Minimizes water interaction

• Stabilizes 3D structure

UV absorbance

The bases absorb the light (delocalized pi electrons in bases).

• Nucleic acids absorb UV light at 260 nm

• Proteins: 280 nm

Chargaff Rules

1. DNA base composition differs among species

2. Same species = same base composition

3. Composition does not change with age or environment

4. number of A=T and number of G=C

DNA helix characteristics

• Right handed

• Antiparallel strands

• bases inside

• sugar-phosphate backbone outside

Major groove and minor groove

major groove is wide while the minor groove is narrow.

B-DNA

The most common DNA form in cells

• right-handed

• 10.5 bp per turn

• bases perpendicular to helix axis

• 3.4 Angstroms between bp

A-DNA

• right-handed

• more compact

• 11 bp per turn

• favored in low water conditions

Z-DNA

• left-handed

• 12 bp per turn

• GC-rich sequences

• occurs in short stretches in cells

Palindromic sequences

Same sequence forward and backward

hairpins and cruciforms

form from inverted repeats. DNA folds back on itself.

Triplex DNA

• three strands

• linked to huntington disease

Tetraplex DNA

• Four strands

• important for telomere stability

RNA structure characteristics

• Mostly single-stranded

• Forms internal base pairing

  • hairpins, loops, stems

RNA helices geometry

A-form geometry

Non-watson-crick base pairs for RNA

G-U, A-A are common

Why RNA forms stable folds

2’-OH enables hydrogen bonding

Metal ions stabilizing RNA

Mg+, K+, Na+

DNA denaturation

Separation of strands

Melting temperature (Tm)

Temperature where 50% is denatured

• the Tm is higher when there is a higher GC content since they have more hydrogen bonds (more stable) than AT.

Hybridization

base pairing between strands from different sources

Blotting techniques

• Southern blot: DNA

• Northern blot: RNA

• Western blot: Protein

Chemical Modifications

• Most common DNA modification: base methylation

• CpG islands:

  • Regions rich in CG

  • Often regulate gene expression

Effects of DNA methylation

• typically inhibits transcription.