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3. Frontiers of Biomedical Engineering:Genetic Engineering
Poziom:
Temat: Edukacja
Professor Mark Saltzman:
This week we're going to
talk about DNA technology and
genetic engineering:
this is Chapter 3 of the book.
Some of this will be familiar
to some of you who've have had
biology in high school or other
places, you know something about
DNA.
In fact, even if you haven't
had a biology class it's hard to
be alive in 2008 and not know
something about DNA;
it's become such an important
part of our lives.
I'm going to ask you to indulge
me while I go back to the
beginning and talk about some
things that you know but I'm
going to go through this pretty
rapidly.
I think the book has a fairly
good description of it so if you
don't pick up everything in the
lecture,
hopefully you've read that
beforehand, and you can go back
to it afterwards and read about
things that didn't make sense.
We'll talk about some
chemistry today,
what DNA molecules are like,
why they have the behavior that
they do,
and you need to understand this
in order to understand how you
manipulate DNA.
So my goal today is to talk
about sort of the basics of the
molecules, their chemistry,
the function of DNA in cells,
sort of basic - the basic side
of that.
Then on Thursday we're going to
start talking about how to
manipulate DNA and get closer to
using it in Biomedical
Engineering.
DNA is a double helix,
you know this,
the double helix was--the
structure of DNA was discovered
about the time that I was born
and so it's been known
throughout your lifetime,
you've always lived with it.
It's really remarkable how
far--how fast we have come from
just knowing the structure of
this molecule to be able to
manipulate it and study it in
great detail.
I want to start by showing you
this cartoon that you already
know about with the structure of
a double helix.
It's a twisted ladder and
there's a couple of things to
notice about this familiar
structure.
One is that there are two
backbones right here in the
light blue, so this would be the
upright parts of the ladder that
are twisted.
Those are two continuous
strands that wind around each
other to form the double helix.
One thing to notice is this
part of the double helix that
we'll call the backbone.
The backbone's on the outside
of the molecule like the upright
struts of a ladder on the
outside of a ladder.
On the inside are the rungs or
the struts that hold the ladder
together.
There are several things
that you'll notice about the
struts in this particular
cartoon.
One is that there's four
different colors and so you can
see red, blue,
yellow,
green here - four different
colors and that's all there are,
there aren't more than four.
That there are two colors per
strut, so what's linking the two
backbones together are two
colored segments that come from
the outside towards the middle,
and that the colors occur only
in certain combinations,
red and green,
yellow and blue,
that's all you see.
You don't see a red and yellow,
you don't see green and blue.
This is a feature of DNA
shown in this cartoon form,
so if you can keep that sort of
schematic in mind,
it makes it a lot easier to
understand the detailed
structure.
That's what I want to do for
the first few minutes of the
lecture here is tell you a
little bit about the details of
the structure and how molecules
fit into this image of DNA
that's already very familiar to
you.
The things that you need to
know are the things that are
really listed on this slide.
You're going to know more
details about it,
they're not really colors,
they're chemicals,
specific chemicals - but the
pattern is the same.
The molecules that really
make up DNA are nucleotides and
DNA is a polymer of nucleotides.
A polymer is just a large
molecule that's made up of
repeated units.
We're familiar with polymers,
plastics in our daily life;
the chairs that you're sitting
on are made of a kind of a
plastic polymer that is
basically an organic chemical
that is cross-linked together.
Cross-linked is not the right
word--that is chemically bonded
with repeat units to make large
molecules so that when you have
a bunch of large molecules
together they have certain
physical properties like the
solid property of the plastic
that you're sitting on.
Nucleic acids,
of which DNA is an example,
are polymers of nucleotides.
So the repeating unit in DNA is
this structure here,
a nucleotide,
which has three different
regions.
There's a sugar,
a five carbon sugar,
which forms the core of the
nucleotide and attached to this
five carbon sugar at specific
positions on the sugar relative
to this oxygen,
which is part of the sugar
ring, is a phosphate group and
an organic base.
When these nucleotides get
polymerized to form a long DNA
molecule they all get
polymerized in exactly the same
way, the chemistry is the same.
The phosphate group of one
nucleotide gets linked to the
sugar group of another
nucleotide and I'm going to show
you that in a few minutes.
So what's going to form the
backbone is this continual link,
phosphate to sugar,
phosphate to sugar,
phosphate to sugar,
all linked together to form one
long,
long molecule.
What's hanging off of the side
of this long molecule that's
formed by polymerizing
nucleotides are - is this base
unit.
It's the phosphate and the
pentose that make up the
backbone - that make up the
upright struts of the ladder and
it's the bases that make up the
connecting struts,
so the bases are the colors.
There are four different bases,
which I'll talk about in a
moment.
We're going to talk about two
different nucleic acid
molecules, two different nucleic
acid polymers,
one is DNA, deoxyribonucleic
acid, the other is RNA,
ribonucleic acid and one of the
differences between the two is
that the pentose,
or the sugar,
that makes up DNA is
deoxyribose shown here,
and the pentose that makes up
RNA is ribose,
shown here so every pentose in
a DNA polymer is deoxyribose.
The phosphate is linked to this
carbon on the pentose,
and notice that there is a
number on this carbon,
it's called - it's the 5'
carbon.
This is a convention that
organic chemist's use when
they're describing molecules
like this.
They'd like to be able to refer
to each carbon separately so
they can talk about reactions
with this molecule,
so they number the carbons.
In this case,
these pentose molecules,
whether it's ribose or
deoxyribose, the carbons are
numbered the same 1',
2', 3', 4', 5',
those are the five carbons that
make up the pentose.
So I could refer to the 4'
carbon and you'd know I'd mean
this one, or the 2' carbon you'd
know I mean this one.
The ones that are important to
us are the 3' carbon and the 5'
carbon.
The reason for that is that the
5' carbon is where the phosphate
is attached.
In nucleotide the phosphate is
always attached to the 5'
carbon.
The reason that 3' is important
is that when you polymerize two
nucleotides together and a third
nucleotide,
and a fourth nucleotide,
when you polymerize nucleotides
together they get polymerized,
the phosphate of one gets
linked to the 3' carbon of
another.
This is important because this
molecule here,
deoxyribose,
is not the same upside down as
it is - it's not symmetrical
upside down and right side up,
it's different because the 5'
carbon's either pointed up or
pointed down.
The nucleotide has a
directionality,
there's an up and a down to it
and it's going to turn out the
chain that's formed by
polymerizing these has a
directionality as well and
that's important in defining the
structure.
These are the pentoses -
remember 5' and 3' because that
orients you with respect to what
direction the molecule is
facing.
Now the bases,
and you don't need to memorize
the structures of these I'm
going - I'm describing the whole
molecule to you in its molecular
detail and then we're going to
simplify it down to a version
that we can talk about more
easily.
To give you all the detail,
there's two classes of bases
that appear here.
One class is called the purines
and they have two ring-like
structures.
There are two of them that are
going to be important to us,
one is adenine and the other is
guanine, shown here.
Because saying adenine takes a
long time and saying guanine
takes a long time we're going to
simplify it by calling adenine
(A) and guanine (G).
The second class is the
pyrimidines and there's three of
those that are important;
uracil, thymine and cytosine
which we're going too simplify
by calling (U),
(T) and (C).
Now remember that there
were only four different colors
in the cartoon of the DNA double
helix that we talked about and I
told you that those colors are
really - represent the bases but
there's five of them here.
There's five because there's
one of these that's particular
to RNA only, that appears in
only RNA, and there's one of
them that appears in only DNA.
The one that appears only in
RNA is (U), the one that appears
only in DNA is (T).
In DNA there's only four
colors, there's only four bases,
(C)(T)(G)(A).
In RNA there's four bases
(A)(G)(U)(C).
So (U) and (T) are
interchangeable in a sense that
(U) appears where (T) would
appear in RNA and (T) appears
where (U) would appear in DNA.
If I drew this altogether
and this is one particular
nucleic acid,
now shown in more detail,
all of the carbons of the
pentose are shown here,
the phosphate is shown,
and a base is shown.
This particular base is (A),
this is ribose.
So this is a monomer or a
single unit from an RNA
molecule, you know that because
it's ribose and it's the
particular molecule that has (A)
as its organic base.
We don't need to really talk
about all the molecular detail
in order to completely describe
a DNA or an RNA molecule because
these structures repeat
themselves.
Every unit in the backbone of
this ladder has the same sugar
unit, the same pentose;
it's either RNA or DNA and has
ribose or deoxyribose,
so you don't need to describe
the whole thing.
You can just say it's RNA or
DNA and you know everything
about the pentose in every
molecule on the chain.
You don't need to say anything
more about the phosphate because
they all have the phosphate and
every set of these is hooked
together in the same way.
The 5' carbon has a phosphate
off of it and that phosphate is
linked to the 3' carbon of the
next one and they all have a
base hanging off the side.
The only thing I need to say in
order to distinguish this
particular part of the chemistry
of a DNA or an RNA molecule is
to say 'it's DNA or its RNA',
and 'what the base is'.
If I told you that 'draw me a
nucleotide from RNA that has
(A)', you could go back to this
picture and you could draw the
whole thing.
You can just talk about it in a
simpler way.
You can say a polymer of DNA,
for example,
is four bases long,
that means it has four of these
repeat units and they go in the
sequence from 5' to 3' of
(A)(G)(T)(G).
If I told you that a DNA
sequence went 5' to 3'
(A)(G)(T)(G) you could draw the
whole thing referring back to
these notes, right?
In fact, you don't have to draw
it this complicated way,
you could just draw it as a
line with (A)(G)(T) and (G)
hanging off of it,
and you would know that that's
a DNA molecule four bases long.
Now what does the line
represent here?
This represents the upright
struts on a ladder that I showed
you before, it represents this
backbone that's shown by -
that's formed by polymerizing
the pentose's together through
phosphate's always going 5' to
3',
5' to 3'.
I could take and draw a line
continually down the molecule
where my finger was touching;
my finger would be touching a
phosphate here,
the 5' carbon,
the 4' carbon,
the 3' carbon,
and the next phosphate.
The backbone is what?
It's phosphate,
carbon, carbon,
carbon, phosphate,
carbon, carbon,
carbon, phosphate and it has
this structure hanging off the
side.
Now DNA is a double helix
and I've only shown you one part
of the helix,
right?
I've shown you one upright
strut and a base hanging off of
it, but it forms a double helix
because complementary strands of
DNA strongly associate with one
another and that's a very stable
structure.
They do that because the bases
can interact with one another in
particular ways,
and this you know about.
This was the famous finding of
Watson and Crick in describing
the structure of DNA.
This is - what this diagram
shows you is--the forces that
hold these individual strands of
DNA into a double stranded form.
The forces occur because of
hydrogen bonding between
complementary pairs of the bases
and the complementary pairs are
adenine and thymine,
(A) and (T) and guanine and
cytosine, (G) and (C).
Now if you read in the
book, you read about where this
figure is shown in the book,
you can understand more about
why these structures line up in
the right way so that the right
molecular elements are together
to form hydrogen bonding pairs
between them.
That's really beyond what I'll
be asking you understand for the
course but you can understand
that if you read it,
I'm sure.
Now remember that (T) only
appears in DNA and (U) appears
in RNA, and so (U) can also form
a hydrogen binding pair with
(A).
The whole structure of a DNA
molecule looks like this,
going back to a more cartoon
version like I showed you before
but adding some detail onto it
now.
There are two upright struts of
the ladder, one shown in blue
here, the other shown black.
They are linked together by
four different colored segments
indicated here not by colors now
but by letters.
It's DNA, so it's (G)(C)(A)(T)
and they always occur in pairs.
Where before it was two colored
pairs now it's two lettered
pairs (G)(C)(T)(A).
The chains are different
now.
They were colored the same,
the backbones were colored the
same in the diagram.
They're colored different here
to indicate one new difference
that you know about now,
and that's that there is an
orientation, there's an up and
down on the chain.
That's due to the asymmetry of
the nucleotide,
that there's a 5' and a 3' end
and the way that they're linked
together.
This blue chain here goes from
5' carbon all along the chain
and there's a 3' carbon left
open at the bottom.
If I wanted to link another
nucleotide to this DNA chain
what would I attach here on the
bottom?
I would attach the phosphate
that's connected to the 5'
carbon of another nucleotide.
I would link this one facing in
this direction I would add onto
this.
The other chain is facing in
the other direction,
the 3' carbon is up,
the 5' carbon is down.
Remember that this molecule,
let's look at the blue one
wouldn't be the same if I turned
it upside down.
It wouldn't be the same if I
turned it upside down because
the carbons - the rings here,
the pentose's would all be
turned over, the chemistry would
look different and the sequence
of bases would look different.
The corresponding half of
the ladder that corresponds to
any given ladder,
let's say the black DNA
molecule that corresponds to the
blue one is not just a mirror
image.
We call it the complement and
each strand of DNA,
each polymer of DNA that you
could make or you could draw has
only one complement and that
complement has the following
features.
One, its chain is oriented in
the opposite direction:
where this one goes 5' to 3',
this one goes 3' to 5'.
It's oriented in the oppose
direction and it has the
complementary base pairs at each
position.
Where there's an (A) here
there's got to be a (T) here,
where there's a (T) here
there's got to be an (A) here,
where there's a (C) here a (G),
a (G), a (C).
That's because you have to
satisfy this base pair matching
in order to have hydrogen
bonding in each of the struts of
the ladder in order to form a
stable structure.
If I'm talking about two
DNA strands and they differ only
in one or two base pairs they
won't be exact complements and
they won't form this double
helix.
This notion of complementary
strands is very important.
It's the way that DNA exists
inside the cells of your body.
It exists in a double stranded
form where every strand is
matched by its complement.
These molecules of DNA,
very long molecules of DNA,
are condensed and packaged
within the nucleus of every cell
in your body.
Every cell in your body has
exactly the same DNA;
that is if I could stretch out
all the DNA and look at the base
pair sequence,
the sequences of bases along
all the DNA in your chromosomes,
they'd be identical in all the
cells.
They'd be different in each of
us and that leads to the
difference in the diversity
between people.
You've heard about the human
genome project,
we'll talk about that a little
bit later.
The goal of that was to take
for a typical human,
or for a typical - in the case
of the human genome project
maybe you're looking at fruit
flies,
you want to look at all the DNA
in a fruit fly,
but to look at the sequence of
base pairs that makes up human
DNA and write them all out;
we'll talk about that later.
This slide shows one
important feature of the
physical chemistry of DNA that
turns out to be very important
for all of the technology that
is built on DNA.
It has to do with the nature of
this complementary binding
between double stranded DNA and
the fidelity of this base pair
matching in forming stable DNA
molecules.
I told you that the fidelity is
very high.
What does that mean?
That only strands that have
this exact complement can form
double stranded DNA.
Because of that you can do
the following experiment,
and it's a simple experiment,
it's simple to understand,
but the concept is very
important so I encourage you to
think about it and make sure you
understand it.
If I took two double stranded
DNA molecules and I exposed them
to certain conditions that
caused them to denature,
that means its native structure
falls apart.
The native structure is this
double stranded structure here
and if I heat it up slightly and
I add some base,
so under slightly basic
conditions, these molecules will
fall apart because you've
created conditions where the
hydrogen bonding is no longer
favorable so they peel apart.
If I had a beaker sitting on
the table here and it contained
a million blue double stranded
DNA molecules and a million red
double stranded DNA molecules
and I heated it up and added a
little base,
I'd soon have four million
individual strands just floating
around in the solution because
I've broken up this hydrogen
bonding and the DNA molecules
fall apart.
That's called denaturing DNA.
That tells you something about
the physical chemistry of the
molecule;
that it's these hydrogen bonds
that hold the double strands and
I can break those down under
certain conditions.
If I then put it back into
its original condition,
lower the heat say,
temperature back to body
temperature and reduce the pH
down to seven again,
the molecules will re-nature.
They will reform their natural
structure, and for DNA that
means forming double helixes.
But they will do that in a very
particular way,
in that only strands that
exactly match will be able to
reform their native structure.
A blue strand here will never
re-nature with a red strand
because their sequences don't
match exactly,
but a complementary blue strand
will always rematch with its
partner.
Now this is the basis of a
physical chemistry process
called hybridization.
It turns out that this is how
we can identify specific DNA
sequences and how we can do
things like DNA fingerprinting,
how we can clone molecules,
DNA molecules from one organism
to another, rely very heavily on
this principle of re-naturation
and hybridization.
Hybridization simply means that
DNA will re-nature and form a
stable double helix only with
its particular match,
only with the hybrid that it is
perfectly complementary too.
That's something about the
physical chemistry of DNA,
what it looks like,
and how it behaves in the
simple sense.
What I want to spend the rest
of the time doing is talking
about some of the biological
properties of DNA.
Again, I know this is something
that's familiar to most of you
and so indulge me just for the
rest of this lecture,
I'll go through it.
I want to try to hit the points
that I think are important to
remember because they're going
to be concepts that come up
again and again throughout the
course,
and I want to make sure that
we're on the same page.
This diagram at the top
here is a very familiar one to
most of you, it's sometimes
called the central dogma of
molecular biology.
It indicates how information
flows in cells and indicates a
lot about the work that a cell
does in maintaining and
recreating itself,
and maintaining its environment.
That is, that the information
needed to operate a cell is
stored in its DNA.
That information gets put into
action through a process,
a biological process called
transcription,
where particular regions of DNA
are transcribed into RNA.
That RNA is made into proteins,
and proteins are the working
molecules of the cell,
they're enzymes,
they're structural molecules,
they're are proteins that exist
in the membrane that allow
things to go in and out of the
cell,
so really the working molecules
are the cell in every sense.
RNA is converted into protein
by a process called translation.
Here's another picture of
it here, showing it in a little
bit more detail,
that you have lots of DNA in
each of the cells in your body
but you're not using all that
DNA at any one time.
Every cell in your body is only
using a fraction of the DNA
that's available to it.
Cells in your pancreas,
for example,
are making the protein insulin.
They're making that because you
need this protein insulin,
it's a hormone,
and it's important for sugar
metabolism in your body.
Those cells in your pancreas
are making insulin.
That means the gene that
encodes insulin,
the sequence of base pairs that
encode insulin.
I'll talk about what that
means, encoding insulin means in
a minute, but there's a gene
that tells your body what
insulin looks like and that gets
transcribed but only in those
cells that make insulin.
It gets converted into a
protein, insulin,
only in those cells that are
able to make the RNA that are
able to express the protein.
Well, it turns out that
proteins are essential in
driving this process too.
In order to have DNA you have
to make DNA and your cells are
continually making DNA inside
your body,
through a process of DNA
synthesis and that synthesis is
occurring because of the
presence of an enzyme,
a protein called DNA polymerase.
In this same way,
this process of transcription
which is occurring in cells
throughout body all the time is
made possible by a protein
called RNA polymerase.
It allows RNA to be made from a
DNA template.
It's not as simple as DNA going
to RNA going to protein,
because proteins need to be
present in order to make these
things happen as well.
Let's talk about DNA
synthesis for a minute.
When a cell divides in your
body, when cells of your
intestine divide,
when cells of your skin divide,
and they're doing this all the
time, in order for a cell to
divide and form two daughter
cells--we'll talk about that
process next week--but in order
for that to happen the parent
cell has to copy all of its DNA
in order to have enough DNA to
pass on to two daughter cells.
It does that through a process
of DNA synthesis.
What happens is the machinery
of the cell, largely this
protein DNA polymerase,
is able to open up the double
stranded DNA,
to denature it locally,
exposing two strands which it
then makes - allows it to make
copies of.
What's shown here is what's
called a replication fork in DNA
that's undergoing synthesis.
The DNA molecule here has been
spread apart,
opening up two single stranded
DNA's which have complementary
base sequences because they were
double stranded DNA.
A new single stranded DNA is
formed on each one of these open
single strands.
So DNA is replicated using one
strand of the DNA as a template.
The result of this process if
this replication went down the
whole length of the DNA would be
to form two identical,
double stranded DNA molecules.
Now the book talks in more
detail about this and you can
read about it.
Polymerase needs a primer and
that turns out to be important.
A primer is a short RNA
sequence or DNA sequence that
gets sort of the process of
replication jump started,
and that's just because of the
biological properties of DNA
polymerase that that primer's
needed.
Synthesis always occurs in
one direction and that makes
sense to you now because you
know there's a directionality
and the chemistry is different
going one way than the other and
this DNA polymerase only works
on the chemistry going in one
direction.
The correct complement is made
because of these principles of
Watson-Crick base pairing that
we talked about before.
It's easy to know what
nucleotide to put in each
position as you're going along
and polymerizing a new molecule.
Because this process occurs
this way, if a parent cell
replicates its DNA and then
passes them along to two
daughter cells,
one of the daughter cells has
one strand from the parent,
the dark blue strand here for
example,
the other daughter cell has the
light blue strand,
the complementary strand,
and each of the daughter cells
has a newly synthesized piece of
DNA.
That's synthesis and that has
to happen in order for cells to
replicate and cell replication
is happening in your body all
the time.
Transcription is also
happening.
Certain segments of DNA are
being converted into RNA,
and whereas in replication,
you have to copy the whole
genome, the whole - all of the
chromosomes, all of the DNA
contained in the chromosomes of
the cell in order to completely
replicate it;
transcription only works on
particular sequences of DNA.
The DNA that encodes the
proteins that are important to
the life of that cell.
A pancreas--cell in the
pancreas, for example,
needs to make insulin and so
the gene for insulin is
transcribed.
Transcription just means
making a single stranded RNA
copy of a sequence of base pairs
in a DNA.
I told you that that's driven
by a protein called RNA
polymerase.
RNA polymerase is smart,
it knows where it needs to go
in order to make the copy of RNA
that's required.
It operates in a similar
fashion to DNA polymerase in
that it denatures locally or
opens up the double stranded
DNA,
but it's different in that it
creates a new polymer from the
DNA template in the language of
RNA,
using RNA nucleotides and not
DNA nucleotides.
The end result of transcription
is not double stranded DNA,
it's single stranded RNA where
the RNA that's produced is
called messenger RNA.
It's the transcribed version of
DNA, and it's the exact
complement of a particular
region of DNA.
Again, more details in your
book if you want to read that.
Well, what I said is not
entirely true.
That used to be the way that we
thought about it.
DNA goes to RNA,
goes to protein,
that's it, and that is the way
it happens in simple organisms
like bacteria.
In complex organisms like
humans there's another step that
we're still only learning about
now.
We know some parts of it,
we don't know all of it.
It's very important in the
biological operation of human
cells and that step is RNA
splicing,
or processing of this RNA,
single stranded RNA that's
produced by transcription.
We're going to talk more about
this as we go through some
specific examples of where RNA
processing is important.
For now, just think about
modifying your picture of this
sort of information flow through
a cell to include another step
that RNA is produced by
transcription from DNA,
double stranded DNA goes to a
single stranded RNA molecule,
and that RNA is processed in
the cell in some way in order to
form messenger RNA.
One of the forms of
processing that happens,
that's very important in human
gene expression is that some of
the sections of the DNA molecule
are not really necessary for
describing the protein.
The regions that are necessary
for describing what the protein
is like are called exons,
the regions that are not are
called introns.
A section of DNA that is
responsible for encoding a gene,
let's say it's the insulin gene
for example,
might be some stretch of DNA on
a certain chromosome inside your
cells, inside the cells of the
pancreas.
If it was directly transcribed
there'd be regions that are
important for making insulin and
regions that are not.
Those regions that are not are
spliced out during RNA
processing to form the mRNA
transcript that's used to make
the protein.
Now there are other kinds
of processing that can happen to
RNA as well, and again,
I said this is really still an
emerging science,
but this is one that's well
known,
and you could imagine that it's
important.
If I want to clone a gene from
a human, if I want to clone the
gene for human insulin,
mean make many copies of the
gene that's responsible for
making insulin,
I need to know whether there
are introns there or not.
If I'm going to make insulin
from this I have to know that
I've got the introns spliced out
correctly.
That's something that will come
up in the lecture tomorrow,
so remember that concept and
that revision of this sort of
classical picture.
I don't want to go through
this in detail because I assume
that you know it,
plus I think it's a little bit
easier to read and have some
time to digest,
but this process of translation
or conversion of messenger RNA
into a protein is a complicated
biological process that's
occurring all the time.
Before, we talked about how do
you know what messenger RNA to
make, how do you know what RNA
to copy from a DNA template?
Well you do that by this
Watson-Crick base pairing,
so I know if I have
(A)(C)(G)(C)(G)(A) I know what
messenger RNA to make from that
because I have to satisfy these
base pairing rules.
It's more complicated in
making protein from an RNA
strand and that complication is
called the genetic code.
You know that messenger RNA is
read in three base units called
codons, and so this particular
piece of messenger RNA is drawn
in this cartoon in three base
pair units.
That's because every three base
pairs describes an amino acid in
a protein.
While there are only four
different bases that make up
either RNA or DNA,
and so the complexity of an RNA
polymer is limited.
It's only got one of four
possible choices at each
position.
There are more than 20 amino
acids that make up the
biological polymers called
proteins,
so there are 20 choices of each
amino acid at a position on a
protein.
Why are there three bases
in a codon?
Because it takes three units
where there's only four choices
at each position to have at
least 20 unique combinations.
How many combinations of codons
are there if there's three bases
and four possibilities at each
base?
4 x 4 x 4--possibilities,
because I could have
(A)(G)(C)(U) here,
(A)(G)(C)(U),
(A)(G)(C)(U) - 4 x 4 = 16.
If I only had two per codon I
wouldn't have enough.
I'd only have 16 possible two
base sequences,
that's not enough to specify
over 20 amino acids.
If I have three,
I have 16 x 4 or 64 possible
choices, way more than enough.
That creates a problem in the
genetic code in that there's 64
possible sequences but there's
only 20 some amino acids,
so each amino acid can be
specified by more than one
codon.
There are combinations to spare.
There are 64 combinations of
three bases and I only need to
describe 20, so there's
combinations to spare.
If I look this table here
shows you how biological
translation takes place whenever
a three base sequence is
identified.
Say it's (G)(C)(U),
that specify an amino acid.
How would I know what amino
acid that is?
Well, I could look up this
table because somebody's figured
it out for you.
(G) in the first position,
(C) in the second position,
(U) in the--(G)(C)(U) right
here is alanine and that's the
protein - that's the amino acid
that's in that position in the
protein.
You could read through this
sequence and you could figure
out what the sequence of amino
acids would be.
The genetic code is said to
be degenerate because I can read
in one direction.
I can read (G)(C)(U) as
alanine, for example,
from this table and if I see a
(G)(C)(U), I know it has to be
alanine.
If I have the protein and I
want to say what does the
messenger RNA that produced that
protein looked like,
I can't go backwards because
there's more than one
possibility for alanine,
right?
There's four of them right here
(G)(C)(U), (G)(C)(C),
(G)(C)(A), (G)(C)(G) so I don't
know exactly what gene that came
from, I can't read backwards.
That has to do with the
statistics of this,
right?
There's just more sequences in
a three unit codon than I need
for the amino acids.
How does translation occur
biologically?
As shown in this cartoon here,
again you don't need to know
the details of this,
but if you're interested in
knowing what's the biological
bases of the genetic code this
is it.
Inside cells in your body there
are special RNA molecules called
transfer RNA.
They are RNA molecules but they
have at some points in their
life span, they have amino acids
attached to them.
For example,
this transfer RNA has a unit
here, (G)(A)(G) at one end of
the transfer RNA molecule.
At the other end,
is attached the amino acid
leucine.
And your cells making transfer
RNA, if they make a transfer RNA
molecule that has (G)(A)(G)
here, they only put leucine at
the other end.
That's the physical basis of
the genetic code because when a
messenger RNA sequence is being
transcribed one base pair - one
codon at a time,
when the sequence (C)(U)(C)
appears in the messenger RNA,
that sequence (C)(U)(C) can
only bind with one particular
three base complement,
it has to be the complement
(G)(A)(G).
This is the codon,
this is the anti-codon,
there's only one anti-codon
that matches this one and that
anti-codon is always - occurs in
a molecule that has leucine
attached to the other side.
Translation occurs by a
special kind of polymerization
where these transfer RNA's
operate by Watson-Crick base
pairing.
They bring into proximity an
amino acid so that instead of
forming a new polymer of a
nucleic acid,
a polymer of an amino acids is
formed.
A polymer of amino acids is a
protein.
Again, I'm just trying to
highlight things you already
know a little bit about,
the book describes the details.
This isn't particularly
important for us to know here,
but that messenger RNA gets
converted into a protein of a
specific composition through a
biological process called
translation is important.
The last thing I want to
talk about today is control of
gene expression.
Control of gene expression is a
very big topic and so I'm going
to show you one cartoon to sort
of tell you that it is a big
topic that's really important.
Why is control of gene
expression important?
Well I talked about it -
earlier I've mentioned several
times all the cells in my body,
all the cells in your body have
essentially the same genomic
chromosomal DNA in their
nucleus.
If you looked at cells in my
pancreas and cells in my brain,
and cells in my skin they all
have the same DNA.
But skin cells and brain cells
and pancreas cells aren't doing
the same things.
They don't look the same,
they don't behave the same,
they don't perform the same
biological functions.
Why?
Because brain cells and
pancreas cells are expressing
different proteins.
All the cells in your body have
the capability of making all the
proteins that you make,
but they're not all made in
every cell.
Only cells of your pancreas
make insulin,
for example.
Only cells in your brain make
the enzymes that produce certain
neuron transmitters that are
responsible for brain function.
How do cells in your brain
know which proteins they ought
to be making,
and how do cells in the
pancreas know which proteins
they ought to be making?
They do that because they can
control the expression of genes.
Gene expression,
for us, will mean the same
thing as production of a
particular protein.
When a gene gets expressed,
that means its protein is
produced.
When we talk about gene
expression than we're talking
about this whole sequence of
events I just described:
transcription,
RNA processing,
translation to make the
protein.
All those things have to happen
in an orderly fashion,
in enough quantity in order for
a particular cell to make a
protein.
To make insulin,
for example,
your cells of your pancreas
have to be transcribing that
gene,
it has to be processed,
has to be translated into the
protein insulin.
But that's not all,
that protein insulin is made in
the form of a long polypeptide
that not - that's not always the
final version of the protein.
In fact, for insulin,
it's not the final version of
the protein that comes out of
translation.
There are more steps that have
to happen correctly in order for
that insulin to become active.
Those steps are called
post-translational
modifications.
It's a long word that just
means other chemistry that
happens on the molecule after
translation.
It turns out that the kinds of
post-translational modifications
that human cells are able to do
are very complicated.
You can do many
post-translational
modifications;
your cells are capable of doing
many post-translational
modifications.
Bacteria, or simple organisms,
are not always capable of that.
Now that's going to be
important when we talk about
making human gene - making human
proteins inside alternate hosts
like bacteria,
that they can't do all the
things that your cells can do.
How is gene expression
controlled?
It can be controlled in a
variety of ways.
The most basic control is by
controlling when transcription
happens.
When transcription happens and
it turns out that there's a
whole biology associated with
this,
including molecules that are
floating around inside your
cells called transcription
factors,
and their job-- they are
molecules that are (that know)
about particular genes and what
some of the sequences and are
able to turn on those genes
inside cells,
to make them transcribe.
It requires RNA processing to
happen smoothly,
so if you can interfere with
RNA processing you can stop a
gene from being expressed.
If you can interfere with any
stage in RNA processing you can
stop a gene from being expressed
and this is a very hot topic in
molecular biology now and human
therapeutics.
You've heard about RNA
interference,
for example,
and that is the process of
stopping this,
to stop a gene from being
expressed.
For example,
a gene that makes a cell
cancerous, I'd like to stop it
from being expressed.
And you could interfere with a
translation by degrading
messenger RNA,
for example.
If you had a way to
specifically chew up all the RNA
molecules that are responsible
for making a particular protein,
you could stop it from being
expressed even though your cell
is trying to make it.
I want you to look at this
picture, read the little bit
about gene - control of gene
expression - that's in the book,
know that it's a big topic,
that we're not going to talk
about it except we're going to
talk about some examples where
control of gene expression can
be exploited in order to treat
diseases,
for example.
So I'll see you on
Thursday.
