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DNA and RNA differ only in a small way
in terms of their structure;
it's just the presence or absence
of that 2’ hydroxyl group.
But that small difference in structure
turns out to have a huge effect on reactivity.
In particular, the 2’ hydroxyl group in RNA
opens up a reaction pathway
for hydrolysis of the phosphodiester bond
that isn't available in DNA.
And in this webcast we want to talk about those differences
and really what we're talking about
is the enzymatic hydrolysis of the phosphodiester group.
These are enzymes that are known as nuclease enzymes,
or phosphodiesterase enzymes.
A typical example is actually found in the toxins
in snake venom in which we have an exonuclease,
meaning that it cleaves nucleic acids
from one end and travels along from one end to the other.
In the case of the snake venom toxin,
it's a 3’ to 5’ direction in which it travels.
It actually digests DNA, not RNA,
so obviously has toxicity to cells.
Some nucleases have specificity,
and these are restriction endonucleases.
That means they cleave in the center of a nucleic acid
with some kind of information specificity such as maybe
as in the case of ribonuclease A,
which we'll talk about in detail
following a particular kind of nucleobase.
Ribonuclease A, for example, cleaves after the pyrimidines,
the C or U.
Ribonuclease A is an RNA cleaving,
ah, nuclease.
It cleaves and takes advantage of this reaction pathway
that's made available by the 2’ hydroxy group.
So what it does, following a pyrimidine,
is cleave the phosphodiester group,
leaving a phosphate group
as well as the hydroxyl group on the 5’ position
of the other side of the nucleic acid.
It involves, then, the addition of water
across that phosphodiester bond.
Let's take a look at the mechanism of how this
ribonuclease A takes advantage of the 2’ hydroxyl group
to open up a reaction pathway
not available to the hydrolysis of DNA.
It's a bifunctional catalysis
mechanism and we've seen many examples of these.
Basically, what that means in this case
is there's a pKa modulation;
the two histidines play off of one another
so that one is going to be a base
and one is going to be an acid;
one's in its protonated form and one is in its neutral form.
Also in the enzyme active site, there's a lysine,
and we'll see that that helps to stabilize
that trigonal bipyramidal intermediate
in the phosphodiester hydrolysis.
The way the mechanism gets started,
right away we'll see how the 2’ hydroxyl group
comes into play.
The most basic position, remember that's
this sp2 position on the imidazole ring of histidone,
histidine, that sp2 hybridized, or ah,
the N2 functional group
as we, we like to refer to that nitrogen as the N2 nitrogen,
is the most basic site.
It's going to deprotonate that hydroxyl group
in a general base catalyzed way,
which will allow it to act as an intramolecular nucleophile
to the phosphodiester phosphorus atom,
and that's what takes place in the first step;
that's the addition step
of the addition/elimination pathway.
Next, what we need to do
is cleave this phosphorus oxygen bond
to generate the 5’ hydroxyl group
on the, ah, exiting side of the, ah, nucleo,
nucleic acid that's cleaved in this, um, in this reaction.
How does that work?
Well, we're going to use the general acid
that's available form the imidazolium cation
on histidine-119
and that's going to be the elimination process,
a β elimination process,
that eliminates this phosphorus oxygen bond,
picking up the proton from that histidine on
the imidazole ring of histidine-119
to make the hydroxyl group.
You can see, then, that that leaves us
with this cyclic phosphodiester,
and in the next step that's going to undergo cleavage
by addition with water.
This negatively charged, this addition,
additional negative charge
in the trigonal bipyramidal intermediate
is stabilized by that lysine-41,
that's the other key thing
that I wanted to bring to your attention.
Alright, let's see how this cyclic phosphate
undergoes hydrolysis with water.
It's actually fairly strained,
quite reactive compared to the normal phosphodiester group.
The way that this happens is the presence of water
in the enzyme active site is activated by a general base;
again, we'll use that most basic N2-type nitrogen
to activate that as a nucleophile.
It'll do an addition step.
Again, that additional negative charge on the, ah,
on this trigonal bipyramidal intermediate
is stabilized by lysine-41.
This, then, turns around and does a cleavage
to leave the phosphomonoester on the 3’ hydroxyl position,
picking up a proton
using histadine-12 as the general acid.
That leaves histidine, ah, 12 in its original form,
this neutral form, and histidine-119
has then left in its protonated form.
We have the phosphomonoester
on the end of the pyrimidine- containing nuc- nucleotide
and the result is we've cleaved the nucleic acid into two.
So to summarize, basically the key difference,
the key pathway that's opened up by that 2’ hydroxyl group
is this intramolecular nucleophile catalyth,
ah, catalyst pathway that makes this
cyclic phosphodiester intermediate.
That pathway is not available to the DNA molecule
and so it's for that reason
that RNA is about a billion or three billion fold
more reactive than DNA.
And the consequence of that is that DNA
is essentially hardwired,
that phosphodiester bond as I mentioned
is extremely resistant.
You can think of DNA as a hard drive,
basically permanent storage of information;
whereas RNA is only temporary carrying information.
It's constantly turned over and so the cell has
capitalized on these very differences in reactivity
to make very different functional use
of these two information carrying molecules.