Molecular Biology Lecture 3
Physical and Chemical Structure of DNA
Structure of DNA subunit (nucleotide):
X-ray Diffraction studies by Rossalind Franklin as well as Watson and Crick showed that:
DNA is helical Ð double stranded helix
Nucleotide bases are stacked
Chemical analysis showed that:
[A] = [T]
[G] = [C]
[A+G] = [T+C]
[purines] = [pyrimidines]
(these ratios only relate to double-stranded DNA. There is also single-stranded DNA in many viruses)
The fundamental structure: (fig. 3-1 and fig 3-5)
10 bases per helical turn (in B-DNA, the predominant form)
right handed helix
two grooves: (these allow the binding of proteins)
wide groove / major groove
narrow groove / minor groove
Also a slightly tighter right handed helix: A-DNA
This has 11 bases per turn (double stranded RNA, dehydrated DNA)
Left- handed helix: Z-DNA
12 bases per turn
Base Ð pairing (fig. 3-2, 3-3, 3-4)
Note that A-T has two H bonds; G-C has three H bonds
Remember the 5Õ ˆ 3Õ polarity of the strands
The strands are anti-parallel
Significance of G+C ratios (or % G+C): for many organisms it is near 50% (for Campylobacter it is 35%. That is, Campy is a very AT Ð rich organism.
Circular DNA
Many bacteria and phage have circular DNA (fig 3-6 and 3-7)
This can exist as:
Relaxed circle
Relaxed nicked circle
Supercoiled (either + [same direction] or Ð [opposite direction])
(-) supercoiling may introduce ÒbubblesÓ and if there are compatible bases Ð get Òstem-loopÓ structures or ÒcruciformÓ structures.
This is especially true if there are Òinverted repeatsÓ or pallindromic sequences present:
ˆ
ATGCÉÉÉÉ.GCAT
TACGÉÉÉÉ.CGTA
§
Enzyme binding sites are often associated with these types of structures.
Important for regulation
Recombination sites
Topoisomerases Ð cut one or both strands of DNA and then either wind or unwind.
DNA Denaturation: (fig 3-8) Melting Curve
Heat double-stranded DNA (native DNA) ˆ It denatures or melts and becomes single-stranded.
This can be measured by spectrophotmetry since the A260 is lower for double stranded DNA than it is for single-stranded DNA.
Tm or T melt is the 50% point
Note that:
á Melting temperature is pretty hot
á It happens pretty fast (narrow temp range)
á Only non-covalent bonds are involved
á Tm is the point at which 1/2 the molecules are denatured
á The higher the G+C content, the higher the Tm
á High pH facilitates the denaturation since it interferes with the base-pairing. High pH ( > 11.3) can be used to denature DNA. [DonÕt use this for RNA though. RNA hydrolyzes at high pH].
á Distilled water can denature DNA. Negatively charged backbone needs to be stabilized with positively charged cations.
DNA Renaturation or DNA Hybridization.
This occurs optimally at 20-25o below the the Tm.
Needs some Na+ or some Mg++ to occur
Curve (fig 3-9) allows you to see if repetitive vs. non-repetitive sequences are involved.
Hybridization techniques are used extensively in the laboratory (fig 3-11)
á ssDNA is attached to a membrane (nitrocellulose)
á then a solution of ÒlabeledÓ ssDNA or RNA is washed over the membrane.
á Wash and visualize by a variety of techniques
o Used in Northern Blots
o Southern Blots
o Microarrays
RNA
Ten times more abundant than DNA
Single-stranded, commonly forms
Stem-loop (hair-pin, cruciform) structures (fig. 3-12)
Often contains ÒmodifiedÓ bases
Hydrolysis of Nucleic Acids
Low pH (less than pH 1) Ð both RNA and DNA hydrolyze (phosphodiester bonds break and the bases break off).
High pH (greater than pH 11) Ð RNA hydrolyzes. DNA will denature but the phosphodieser backbone remains intact.
Nucleases
DNase, RNase
Exonuclease, Endonuclease, Restriction endonuclease
DNA Sequencing Ð Sanger Method (Dideoxy chain termination technique)
Remember that polymerases only catalyze DNA synthesis 5Õˆ3Õ, and new nucleotides are added to a free 3Õ-OH group.
This technique uses dideoxynucleotides to terminate a proportion of DNA synthesis reactions.
Four separate reactions are run. You have to run a separate reaction for each of the four nucleotides.
Cover figures 3-15, 3-16, 3-17.