talk about the structure of DNA and RNA
Warming up the brain: Nucleic acids are made up of nucleotides, consisting of
bases (purines and pyrimidines), as you probably recall from
your genetics or cell biology class, sugars (ribose or deoxyribose),
and a phosphate backbone.
Remember that we have some rules, called "Watson-Crick" base pairing, by
which adenylate nucleotides can hydrogen bond to thymidylate nucleotides (or
uridylate in RNA), while guanylate nucleotides hydrogen bond to cytidylate nucleotides.
A pairs with T (or U)
Is this all starting to come back to you now? Let's find out.
about the bases
Stop me if you've heard this one...
A guy walks into a bar and says "My name's Chargaff, and 22% of my DNA is "A"
nucleotides. I'll bet anyone that they can't guess what percentage of my DNA is "C"
nucleotides!" You say "I'm thirsty, so I'll take that bet!" and then
Yes, it can be done! Erwin explains, we have double stranded DNA genomes, so if 22%
is "A", then there must also be 22% "T", because every "A"
base will be paired with a "T" base. You with me? So 22%+22%=44% is the
percentage of the DNA that is either "A" or "T". That implies
that the percentage that is "G" or "C" must be whatever is left,
or 100%-44%=56%. Every "G" must be base paired with a "C" and
every "C" must be base paired with a "G", so exactly half of
that 56% must be "C" bases. That is, 28% are "C" bases and 28%
are "G" bases.
Here's a photo gallery (Click for larger images):
red = oxygen, blue
= nitrogen, white = hydrogen, gray = carbon.
What atom does the amber color represent?
The nucleotide bases make up the core
of the double helix, as you can see in the picture below.
This snapshot comes from a site you'll probably want to investigate, an "Interactive
Animated Nonlinear Tutorial"
by Eric Martz, from the Department of Microbiology at the University of Massachusetts-Amherst.
Here's another good site to visit, to learn about the Chime plug-in,
and to study the overall structure of DNA. Chime is pronounced with a hard "K"
sound as in "kind", not a "Ch" sound as in "chair."
You can develop a real "feel" for molecules if you familiarize yourself
with the shareware RasMol (RasMac) program. With this program, you can
inspect crystallographic structures downloaded from Brookhaven National Labs, turning
the molecules on the screen so you can see them from every side and angle. Downloading
instructions are available on
the Web, as are instructions for finding molecules to play with.
If you have the Chime plug-in working, you may be able to see the following two examples,
generated by GLACTONE (http://chemistry.gsu.edu/glactone/). You may also download them directly and use
An AT base pair
hydrogen bonding come to pass?
Well, suppose this is a cherry, and you're going to make chocolate cupcakes with
cherries on top. You make the cake mix, fill the little cupcake holders and bake
the cupcakes. Then you put a cherry on top of each, and whip up a batch of chocolate
icing. Here is one, ready to cover with frosting!
Here's one that was covered well, in fact it was so evenly covered with frosting
that you can no longer see the cherry!
Then, an interesting thing happens. On some of the cupcakes, the chocolate icing
is very thin. It dribbles down onto the cake, leaving the cherry somewhat visible
through the frosting.
It is almost as if the cupcake and the cherry are fighting for the frosting, and
the cupcake is winning!
In fact, sometimes the frosting gets so thin, that there's nothing left to hold the
cherry in place, so it pops out, leaving the frosting still stuck to the cake.
Hmmm... What does this make us think of? Why polar covalent bonds, of course!
You see, some atoms are more electronegative
than others. Oxygen is more electronegative than hydrogen, so in an -OH group, the
oxygen takes more than its fair share of electrons. That's just like the cupcake
taking more than its fair share of frosting. The electrons get very thinly distributed
over the hydrogen and get more thickly distributed over the oxygen.
That gives a partial negative charge to the oxygen and a partial positive charge
to the hydrogen. Why? Because the electron is charged, and if more of it is distributed
in one place, that place will get a bit of charge.
Nitrogen can play the same trick, because it is also more electronegative than hydrogen.
On the other hand, carbon and hydrogen are about the same in electronegativity, so
they share the electrons pretty fairly. There will not be a partial charge on the
carbon, because the electrons are distributed evenly in the bond. The carbon-hydrogen
bond reminds us of the well-frosted cake - all neutrally distributed:
On the other hand, the oxygen-hydrogen and nitrogen-hydrogen bonds remind us of the
thinly-frosted cake, and the thin frosting leads to a "dipole moment",
or partial charge:
difference between DNA and RNA?
DNA contains the sugar deoxyribose while RNA is made with the sugar ribose. It's
just a matter of a single 2' hydroxyl, which deoxyribose doesn't have, and ribose
does have. Of course, you all remember that RNA uses the base uracil instead of thymine
Cytosine naturally has a high rate of deamination to give uracil
Cytosine deamination (i.e. water attacks!)
Uracil in the DNA is a big no no,
and there are specific enzymes called uracil N-glycosylases (from the gene called
ung, about which we'll have much more to say in a later lecture) that excises
the offending deoxyuridylate nucleotide so that it can be replaced. If the uracil
had arisin by deamination, then what will be the nucleotide base across from it?
There will be a G nucleotide across from it, if the mutation just occurred. That's
because the G was paired with the C that deaminated to a U. On the other hand, if
there is a round of DNA replication before the uracil N-glycosylase arrives on the
scene, then there will be an A nucleotide across from the U. That's because the U
will have had a chance to be a template in DNA replication, and U base pairs to A,
If you're an organism that doesn't want
to end up looking like a Teenage
Mutant Ninja Turtle (who as
you may recall, were suffering from the effects of a "retromutagen" that
made them behave like adolescent boys), then you should keep a sharp eye out for
deoxyuridylate nucleotides. The dU should be excised rapidly and replaced with a
C, so that these deamination events do not become "fixed" as a mutation.
Some types of mutations change a pyrimidine
to a different pyrimidine, or a purine to a different purine. We call these transition mutations. If a purine is mutated to a pyrimidine, then
it is a transversion
mutation. So, for example, a mutation
of A to T or C to A would be what? Right! A transversion, and a mutation of A to
G or T to G would be a transition.
Sometimes deoxycytosine is methylated on its "5 position," so what would
happen to the coding content of deoxy-5-methyl-cytosine if it were unlucky enough
to be naturally deaminated?
Deamination of 5-methyl cytosine gives you
Do you know my name?
So you see the problem...the 5-methyl cytosine is deaminated to thymidine. The new
thymidine looks like any other thymidine - it's a mutation! A transition mutation,
because it is a pyrimidine changed to another pyrimidine.
Perhaps that is why there are so few CG dinucleotides in mammalian genomes. CG dinucleotides
are frequently methylated on the C base, so CG may frequently mutate to TG, leaving
CG "under represented". In fact, CG dinucleotides are sometimes associated
with regulatory regions of genes, and we call them "CG islands" because
they are so rare.
about the sugars
Now let's look at the sugar
component of nucleic acids. Remember that ribose and which is deoxyribose?
There is a 5' end
and a 3' end to a nucleic acid. The 5' end frequently has
a phosphate attached, while the 3' end is typically a hydroxyl group. A single strand
of DNA has a "polarity" or "directionality."
It isn't like a piece of string, in which you cannot distinguish one end from the
DNA vs. RNA sugars
Deoxyribose with thymine base
Ribose with uracil base
Study the phosphate at the 5' end
click for larger image
Study the hydroxyl at the 3' end
click for larger image
Synthesize? Degrade? Sit and wait? How
does an enzyme like DNA polymerase Klenow Fragment know what to do next? Well, there
are some general rules of conduct that these enzymes learn in school, and you can
learn them too.
rules of conduct for Klenow and T4 DNA polymerases
1. Remember your base
pairing rules: G goes with C and A goes with T.
2. The 5' ends are strictly
off limits, unless you have your holoenzyme license (and for your information, you
3. There will be no
synthesis without a free 3' end, unless you have your RNA polymerase license (and
for your information, you don't!)
4. There will be no
degradation without a free 3' end, unless you have your endonuclease license (and
for your information, you don't!)
5. There will be no
synthesis without an underlying template, unless you have your terminal transferase
license (and for your information, you don't!). Excess nucleotide substrates is NOT
accepted as an excuse for untemplated additions to the 3' end.
6. Under no circumstances
may you make a synthetic addition to the 5' end (even holoenzymes are not permitted
to do that!). Having a template or substrate available is not an excuse for 3' to
7. There will be no
reconstruction of a broken phosphodiester bond, unless you have your ligase license
(and for your information, you don't!). If you are synthesizing DNA and run into
an obstruction on your template, you must stop and leave the nick unrepaired. You
may not excise the 5' nucleotide that is obstructing your path (see rule 2).
8. If you have no remaining
template, then you must excise the nucleotide at the 3' end (and don't be tempted
to break rule 5!). (repeat rule 8 until it does not apply).
9. If you have been
provided with a free 3' end, a template, and a substrate molecule that is correct,
you must add that nucleotide to the growing end of the strand (i.e. to the 3' end.)
10. If you have a free
3' end and a template, but after waiting for the appropriate number of milliseconds
you are still missing the appropriate nucleotide substrate for the next synthetic
step, you may go back and remove the one preceding nucleotide. Either of rules 9
or 10 may apply thereafter.