molicular biology
TRANSCRIPT
Types of Molecules
• DNA: code for genetic information
• RNA– mRNA intermediates in protein synthesis– rRNA carry out protein synthesis– tRNA translate info on mRNA into a.a.
• Gene
www.freelivedoctor.com
Nucleotide Components
• Bases + sugar + phosphate• Bases
– purine• adenine
• guanine
– pyrimidines• thymine
• cytosine
• uracilwww.freelivedoctor.com
Bases
N N
NN
NH2
N N
NN
O
NH2
N
N
O
O
CH3
N
N
O
NH2
N
N
O
O
Adenine (A)
Guanine (G)
Purines Pyrimidines
Thymine (T) (DNA only)
Cytosine (C)
Uracil (U) (RNA only)
www.freelivedoctor.com
Nucleotide Components
• Sugars– ribose
– Deoxyribose
– Rings numbered using primes
• C1 = 1’ etc
• PO43-
OH
OHOH
OHOH2C
OH
HOH
OHOH2C
ribose (RNA)
2-deoxyribose (DNA)
www.freelivedoctor.com
Nucleotide Structure
• Base-sugar-phosphate
• Base attached to C1 of sugar
• Phosphate attached to C5 of sugar
• Usually nucleotides are triphosphates, but can be mono or di
OH P O
O
O
P OO
O
P O
O
O
N
O
N
N
OH
OH
N
NH2
OH P O
O
O
P OO
O
P O
O
O OH
OH
N
N
O
O
CH3
Adenosine Triphosphate (ATP) (a ribonucleotide)
Thymidine Triphosphate (TTP) (a deoxyribonucleotide)
www.freelivedoctor.com
Nucleic Acid, a polymer of nucleotides
www.freelivedoctor.com
Nucleic Acids
• Polymer of nucleotides
• Phosphodiester linkage – 3’ of one sugar to 5’ of other– vectorial
• Sequence read 5’ - 3’
• Previous slide is C-A-A
• Next slide (a cartoon drawing) is A-T-G-C
www.freelivedoctor.com
Phosphodiester Bond
www.freelivedoctor.com
Chain elongation
• 3’ hydroxyl on chain has unshared pair on oxygen– attacks phosphorus on first phosphate of new nucleotide– Nucleophilic attack
• Driven by hydrolysis of PPi
OH P O
O
O
P OHO
O
O P OH
O
O
pyrophosphate
2
phosphate
www.freelivedoctor.com
Chain elongation
H
OO
N N
NN
O
N
P
O
O
OR
OH
OH P O
O
O
P OO
O
P O
O
O
N
O
N
N
H
OH
N
NH2
H
OO N N
NN
O
N
P
O
O
OR
O
PO
O
O N
ON
N
H
OH
N
NH2OH P O
O
O
P OHO
O
+
chain
nucleotide
3- OH
+
longer chainpyrophosphate
www.freelivedoctor.com
Base pairs
– Bases are planar• sp2 hybridization• Bases nonpolar
– In Nucleic acids, there is base pairing– Complementarity
• Purines always base-pair with pyrimidines– Cytosine hydrogen bonds with guanine– Thymine hydrogen bonds with adenine or uracil
• Due to distancing and proximity of unshared pairs and hydrogens
www.freelivedoctor.com
GC base pair
NN
N
NH
O
N HR
H
Guanine
NN
O
N
H
H
R
-- -- --
-- -- --
-- -- --
cytosine
3 H- bonds
www.freelivedoctor.com
AT base Pair
N
N
N
N
N H
H
R
adenine
NNH
O
O
R
-- -- --
-- -- -- --
thymine
2 H-bonds
www.freelivedoctor.com
A-U base Pair (RNA)
N
N
N
N
N H
RNN
O
O
H R
H
adenine uracil
2 H bonds
www.freelivedoctor.com
Base Pairs (Cartoon drawing)
www.freelivedoctor.com
DNA structure
• Double helix
www.freelivedoctor.com
DNA structure
• B-DNA is most common• Antiparallel• bases inside
– Hydrophobic– Perpendicular to helix
• stands complementary– Very important for information transfer– Each strand a template for the other.
• right handed• major and minor groove
www.freelivedoctor.com
Other forms of DNA
• Z - DNA left handed; structure not fully understood
• A- DNA; tighter helix
www.freelivedoctor.com
DNA TypesB- DNA (most common)
www.freelivedoctor.com
A-DNA (supercoiled)
www.freelivedoctor.com
Z-DNA
www.freelivedoctor.com
Look down Axis
www.freelivedoctor.com
Sequence dependent structures
• palindromes
• hairpins
• inverted repeats
• mirror repeats
www.freelivedoctor.com
RNA
• codes for protein
• no consistent secondary structure
• single stranded
• Ribose instead of deoxyribose
• Thymine (T) replaced by Uracil
www.freelivedoctor.com
RNA
O
OHOH
CH2OO P
OO
O
N
OH
CH2O
O P
OO
ON
OH
CH2OO P
OO
O P O
O
O
O N
OH
N
NN
NH2
CH2
N
O
O
N
NN
O
NH2
N
N
O
NH2
5' end
A
U
G
C
3 ' end
www.freelivedoctor.com
Transfer RNA
www.freelivedoctor.com
Instability of RNA
• RNA unstable due to reactivity of 2’ hydroxyl on ribose
• Very labile
• DNA extremely stable
www.freelivedoctor.com
Nucleic Acid Chemistry
• Denaturation– unzip by heating– reannealing– CoT curves: more GC=higher temp
• Uses of DNA to find sequences– probes– forensic
www.freelivedoctor.com
DNA probes
• Take DNA from crime scene
• DNA from suspect, random other person etc
• See what matches
www.freelivedoctor.com
Nucleic Acid Chemistry
• Mutations– changes in
sequence/structure
– nonenzymatic changes• thymine dimers
• deamination
• oxidative damage
www.freelivedoctor.com
Nucleic Acid Chemistry
• Sequencing
• PCR
• Southern Blotting
• Probes
www.freelivedoctor.com
DNA sequencing, Sanger dideoxy method
• 4 samples• Each gets template and nucleotides
– One dideoxy nucleotide– Has no 3’ OH so chain can’t elongate
• Sample 1– ddTTP…stops at T
• Sample 2– ddATP…stops at A
• Sample 3– ddCTP…stops at C
• Sample 4– ddGTP…stops at G
• Run on Gel– Read from bottom up
www.freelivedoctor.com
Sample Sanger Gel
www.freelivedoctor.com