Post on 04-Feb-2022
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Nucleotides and nucleic acids
Lecture 5
Dr. Mamoun Ahram
Nucleic acids
• Monomers
• Polymers
Chemical composition and bonds
DNA vs. RNA
Nitrogenous bases
Other nucleotides
• Xanthine, hypoxanthine and uric acid
– intermediates in purine metabolism)
• N6-methyl adenine
• 5-methyl-cytosine and N4-methyl cytosine
• pseudouracil
– has the ribose attached to C5 instead of N1 of uracil
– Pseudouridine
• 1,3,7-trimethylxanthine (caffeine)
N6-methyl
adenineC5-methyl
cytosineN4-methyl
cytosine
Nucleotides are acidic
Nucleotides vs. Nucleosides
Nucleic acid polymer
A new era
DNA structure
• A double helix
• Backbone vs. side chains
• Antiparallel
• Specific base-pairing
– Chargaff's rules
• Stable
• Flexible
• Groovings
• Stability vs. flexibility
• Chargaff's rules
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Chemical forces
• Ionic interactions
• Hydrogen bonds
• van der Waals interactions
• Hydrophobic interactions (Base stacking)
• Both van der Waals and hydrophobic interactions drive the formation of DNA (and RNA) double helices
B-DNA
Other forms
Chromosomal packaging
• 2 meters of DNA/cell
– 40 km of thread in a tennis ball!
• How?
– Histones
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What is a chromatin?
• Chromatin = DNA molecule + proteins
• The proteins that bind to the DNA:
– histones (H1, H2A, H2B, H3, and H4)
– nonhistone chromosomal proteins
Nucleosomes
Chromatosome
• H1 is bound to the DNA forming with the octamer and wrapped DNA a chromatosome
DNA-histone interaction
• Histones are positively charged
– Interaction
– Charge neutralization
RNA
• Vs. DNA
• Secondary structures
Molecular processes
Types of RNA
Messenger RNA (mRNA)
• Localization
• Modification
• Variable size
Types of RNA
Ribosomal RNA (rRNA) • Ribosomal RNA molecules comprise 65 to 70% of the mass of the
ribosome
• The rRNA maintains the structure of the ribosome and provides sites for the binding of mRNA and protein synthesis
Types of RNA
Transfer RNA (tRNA)
• tRNA is a ~75 base molecule that carries the amino acids, and transfers them to the growing protein
• tRNAs have a common tertiary structure
Modified nucleotides in tRNA
• tRNAs contain modified bases
Other RNA molecules
• Small nuclear RNA (snRNA)
– RNA processing
• MicroRNA (miRNA)
– Natural
– translation regulation
• Small interfering RNA (siRNA)
– Synthetic
– Translation regulation
Light absorbance of nucleic acids
• Aromatic pyrimidines and purines can absorb UV light
• The peak absorbance is at 260 nm wavelength
The unit of absorbance
• The absorbance of 260 nm (A260) is constant – dsDNA: A260 of 1.0 = 50
ug/ml
– ssDNA: of 1.0 = 30 ug/ml
– ssRNA: A260 of 1.0 =40 ug/ml
Exercise
What is the concentration of a double stranded DNA sample diluted at 1:10 and the A260 is 0.1?
DNA concentration = 0.1 x 10 x 50 µg/ml
= 50 µg /ml
Denaturation-renaturation
Observation of denaturation
Factors influencing Tm
• G·C pairs
• pH
• Salt and ion concentration
• Destabilizing agents (alkaline solutions, formamide, urea)
G-C content