gene regulation lecture 1

8/7/2019 Gene Regulation Lecture 1

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Gene expression

Genome

Transcriptome

Proteome


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DNA is the genetic material of bacteria (Griffith, 1928)

Bacterial transformation provided the first proof that DNA isthe genetic material. Genetic properties can be transferredfrom one bacterial strain to another by extracting DNA fromthe first strain and adding it to the second strain.


3/15

Avery 1944


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DNA structure

Polynucleotide chains havenitrogenous bases linked to a sugar-phosphate backbone

Positions on the ribose ring are

described with a prime () todistinguish them.

DNA has a deoxyribose sugar (2-H);RNA has a ribose sugar (2-OH).

DNA contains adenine, guanine,cytidine, and thymine; RNA has uracilinstead of thymine


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DNA structure

Nucleoside - purine or pyrimidinebase linked to position 1 of apentose sugar.

Nucleotide - a nucleoside linked to aphosphate group on either the 5 or

3 position of the (deoxy)ribose.

Successive sugar residues arejoined by a phosphate groupbetween the 3 position of one sugarand the 5 position of the next sugar.

One end of the chain (conventionallythe left) has a free 5 end and the

other end has a free 3 end.


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DNA is a double helix

The B-form of DNA is a doublehelix consisting of twopolynucleotide chains that runantiparallel.

The nitrogenous bases of eachchain are flat purine orpyrimidine rings that faceinwards and pair with oneanother by hydrogen bonding to

form A-T or G-C pairs only.


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Forces that control nucleic acid structure

1. Sugar-phosphate backboneis negatively charged;negative charge neutralizedby metal ions and positivelycharged proteins

2. Base-pairing (hydrogenbonding)

3. Base-stacking (hydrophobic

interactions)


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9/15

DNA can be denatured and renatured

Forces that stabilize DNA can bedisrupted by:

1.Heat

2.Low salt concentration

3.Change in pH


10/15

DNA denaturation or melting

Followed by change in optical density; bases adsorb light in the UVrange (260 nm)

Adsorption of DNA is 40% less than that of free nucleotides due tostacking 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 temperaturerange for denaturation

Tm increases approx. 0.4C for every 1% GC content

40% G.C - 87C60% G.C - 95C


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Renaturation

Requirements:1. Temperature must be 20-25C 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


12/15

The ability of two single-strandednucleic acid preparations tohybridize is a measure of theircomplementarity.


13/15

The diameter of the double helix is 20 ,and there is a complete turn every 34 ,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


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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 vivoZ 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


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B, A, Z forms of DNA

gene regulation lecture 1

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