gene regulation lecture 1
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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.
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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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DNA can be denatured and renatured
Forces that stabilize DNA can bedisrupted by:
1.Heat
2.Low salt concentration
3.Change in pH
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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
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The ability of two single-strandednucleic acid preparations tohybridize is a measure of theircomplementarity.
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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
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