Gene expression

Genome

Transcriptome

Proteome

DNA is the genetic material of bacteria (Griffith, 1928) Bacterial transformation provided the first proof that DNA is the genetic material. Genetic properties can be transferred from one bacterial strain to another by extracting DNA from the first strain and adding it to the second strain.

Avery 1944

DNA structure
Polynucleotide chains have nitrogenous bases linked to a sugarphosphate backbone Positions on the ribose ring are described with a prime (´) to distinguish them. DNA has a deoxyribose sugar (2´-H); RNA has a ribose sugar (2´-OH). DNA contains adenine, guanine, cytidine, and thymine; RNA has uracil instead of thymine

DNA structure
Nucleoside - purine or pyrimidine base linked to position 1 of a pentose sugar. Nucleotide - a nucleoside linked to a phosphate group on either the 5´ or 3´ position of the (deoxy)ribose. Successive sugar residues are joined by a phosphate group between the 3´ position of one sugar and the 5´ position of the next sugar. One end of the chain (conventionally the left) has a free 5´ end and the other end has a free 3´ end.

DNA is a double helix
The B-form of DNA is a double helix consisting of two polynucleotide chains that run antiparallel. The nitrogenous bases of each chain are flat purine or pyrimidine rings that face inwards and pair with one another by hydrogen bonding to form A-T or G-C pairs only.

Forces that control nucleic acid structure
1. Sugar-phosphate backbone is negatively charged; negative charge neutralized by metal ions and positively charged proteins Base-pairing (hydrogen bonding) Base-stacking (hydrophobic interactions)

2.

3.

DNA can be denatured and renatured
Forces that stabilize DNA can be disrupted by: 1.Heat 2.Low salt concentration 3.Change in pH

DNA denaturation or melting
Followed by change in optical density; bases adsorb light in the UV range (260 nm) Adsorption of DNA is 40% less than that of free nucleotides due to stacking of bases; this effect is called HYPORCHROMIC EFFECT Denaturation can be followed by HYPERCHROMICITY The Tm or the melting temperature is the midpoint of the temperature range for denaturation Tm increases approx. 0.4°C for every 1% GC content 40% G.C - 87°C 60% G.C - 95°C

Renaturation
Requirements: 1. Temperature must be 20-25°C below the Tm 2. Salt concentration must be high (0.15M - 0.5M)

Slow process: Rate limiting step is the precise collision between complementary strands Detected by: 1. Solution hybridization a) follow change in optical density OR b) follow reaction with radioactive label (separate ds and ss DNA OR degrade ssDNA) 2. Filter hybridization

The ability of two single-stranded nucleic acid preparations to hybridize is a measure of their complementarity.

The diameter of the double helix is 20 Å, and there is a complete turn every 34 Å, with 10 base pairs per turn. The double helix forms a major (wide) groove and a minor (narrow) groove. Most DNA sequence specific proteins bind through the major groove

Alternative double helix structure
Three major forms: A, B, Z ds RNA and DNA/RNA duplexes are in the ‘A’ conformation Z DNA may occur transiently in vivo Z conformation is dependent on: 1. Sequence poly d(GC), poly d(AC) 2. Base and backbone substitution - poly d(Gme5C) 3. High ionic strength 4. Negative supercoiling

B, A, Z forms of DNA