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OK.  And here we are in the
molecular biology section.

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And the goal of this section,
as Professor Jacks started to tell

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you during the Genetics module and
Professor Baker told you at the

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beginning of last lecture is try to
link together in molecular terms the

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question of genotype and the
question of phenotype.

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And we presented to you this notion
that goes by the ponderous name of

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the central dogma that the link
between genotype and phenotype is

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related to DNA as a genetic material
that then proceeds to transmit its

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information to a final outcome,
which is very often a protein,

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through an RNA intermediate.
And the point of these molecular

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biology lectures is to tell you
about the molecular biology,

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and then at the end to try to bring
together this genotype and phenotype

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in molecular terms.
Now, last lecture you talked about

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DNA replication,
DNA as the genetic material required

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to be replicated faithfully and
accurately so it can transmit its

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information to the next generation.
Professor Baker I know stressed the

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requirement for accurate replication,
but she did not do one part of this

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lecture that I want to spend a
couple of minutes now discussing

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with you.  And that is the question
of DNA repair.

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So there are two types of DNA repair.

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Actually, three types that I want
to talk to you about.

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And the first pertains to the
accuracy of the DNA polymerase that

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replicates the DNA.  So
DNA polymerase --

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-- three, or the DNA polymerase that

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replicates the DNA makes mistakes.
It puts in the wrong nucleotide.

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It puts in the wrong base.
And it does so about one in ten to

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the fifth bases.
OK?  So one in a hundred thousand

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bases is wrong.
Now, if you think about the fact

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that there are more than ten to the
ninth bases in a human genome,

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every cell cycle that translates to
ten thousand or so mistakes,

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that's a lot of changes in the DNA.
That's not a very faithful kind of

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DNA replication.
So this has been selected against

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evolutionarily.
And there is a mechanism that's

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called proofreading --

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That allows this high error rate to
be corrected.  And it's actually

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very cleaver.  So this DNA
polymerase has what is called an

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exonuclease activity.
Exo meaning out.  Nuclease meaning

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to break down nucleic acids.
And this exonuclease proceeds from

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the 3 prime to the 5 prime direction,
the 3 prime nucleotide being the one

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that was added last as
you should now know.

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And so what DNA polymerase does as
it is replicating is it kind of

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feels whether or not the double
helix has reformed in a smooth way.

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And if it feels that there is a
bubble there, a bubble where the two

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bases, actually look at me rather
than the diagram.

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I think it's easier.
I can do it better with my hands.

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Where you've got a nice smooth
helix, if there is a mismatched

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nucleotide, the bases do not pair,
there will be a bubble.

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OK?  There will be a bubble in the
helix or a space in the helix.

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The two strands will not be joined
together.  And the DNA polymerase

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can sense this and it can go back
and it excises the wrong nucleotide

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and puts in the correct one.
OK?  This is called proofreading.

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And it's extremely necessary and
it's actually very good.

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And what it does is to decrease the
error rate to one in ten

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to the ninth bases.
OK?  So you get four orders of

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magnitude improvement in the
accuracy of DNA replication.

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Now, there is another set of things
that can go wrong.

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And these actually fall under the
heading of mutagens.

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Mutagens, as Professor Jacks
mentioned to you,

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being agents which change the base
sequence of the DNA once

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the DNA is there.
And these can either be chemical or

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these can be ionizing radiation.
And in those cases also the helix

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gets changed because the wrong base
gets put in.  No,

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not because the wrong base gets put
in.  But because there is a chemical

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reaction which might modify a base,
which might, for example, covalently

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link two bases.
thymine for example.

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If two thymines are sitting next to
one another in the helix,

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ultraviolet light is very good at
cross linking those.

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And you now have something called a
thymine dimer.

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And that is very bad because that
is not a normal base sequence.

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And when replication time comes
along that DNA helix is abnormal and

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the replication machinery doesn't
know what to do about it,

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and that can lead to all sorts of
problems and to mutations.

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So there are mechanisms that can
get rid of abnormal bases.

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So mutagens can chemically,
actually, maybe not say chemically.

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Let me just say change bases.  They
change base structure either to

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something that looks like another
normal new base or to something that

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looks abnormal.
And there are two mechanisms to get

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rid of this.  One is called excision
repair and the other is called

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mismatch repair.
I have them written in the reverse

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order than is on this diagram from
your book.  In mismatch repair there

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is one nucleotide that looks normal,
but it's different.  It doesn't

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match the, usually it looks normal.
It doesn't match the one opposite

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to it.  And in that case the repair
machinery can go in and remove the

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abnormal or the mismatched
nucleotide, and there's another

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enzyme that will go and correct it.
In excision repair,

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one very often, excision repair
occurs when, for example,

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two nucleotides have become
covalently linked to one another,

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and the one strand of the helix is
just a mess.  And there is an enzyme,

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or enzyme complex that will go in
and actually excise a little chunk

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of the helix.  And then another
enzyme will come in and fill in the

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gap so that you get the
helix repaired.

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Now, the challenge in this,
and you may be asking yourselves

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this, is how does this repair
machinery know which the correct

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strand was?  In the case of
proofreading it's very interesting

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because initially after replication
the newly synthesized DNA strand is

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not modified.  It's just a normal
nucleotide polymer.

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However, the template strand,
the template strands, the parental

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strands over time become
chemically modified.

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The bases actually get,
especially adenine gets some methyl

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groups added to it.
And this  is different than the

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newly synthesized with doesn't have
these methyl groups.

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And so the polymerase knows which
strand is the old strand and the

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correct one and which is the new
strand and the incorrect one.

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In the case of excision and
mismatch repair,

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that's sometimes not clear.
Where you've got these thymine

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dimmers, these Ts that are joined
together then that's clearly the

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wrong, that's clearly wrong.
OK?  The enzymatic machinery can

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take that out and copy the other
strand.  Sometimes,

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though, if you just have a chemical
conversion of one base to another,

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the repair machinery does not know
which strand is the correct and

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which isn't.  And that's when you'll
get mutations fixed in the DNA

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because at replication you really
may get the changing,

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you may get the incorrect,
you may not get the correct base

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repairs.
And then that incorrect base will be

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passed on through the next
generation.  OK.

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So this is a very rapid zip through
DNA repair that I wanted you to be

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able to think about.
I want to move onto the next step

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in the transmission of information
from gene to final product today.

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And I want to talk to you about the
generation of RNA.

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And so let us begin with a quiz.
And I have for you a new incentive

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to pay attention,
a new prize that you can use to

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think about the conversion of
potential to kinetic energy,

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and also you can use to amuse
yourself when you're downloading

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very poor, when you're downloading
things from the Internet and have

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nothing better to do.
I can usually get this right across

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the room.  There you go.
You can also use it to think about

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the nature of amphibians,
they're nice flying frogs.

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OK.  So let us pose the question
here, what is RNA?

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And you've had some of this on a

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problem set, but you really need to
know what I'm talking about.

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This is a ribonucleotide.  How do I
know that this is a ribonucleotide?

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Think about it.  You can put your
hands up, but I want everyone to

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think about it.
OK.  And you need to identify the

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precise chemical group,
please, that tells me.  I saw you

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two first, so yes.

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What does the lower right mean?
Give me a name.  It's the?  There's

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a number there.
The?  Ah, we have a discrepancy of

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opinion here.  Someone says it's a 3
prime hydroxyl on the ribose and

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someone says it's the 2 prime
hydroxyl on the ribose.

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Let's take a vote.
Who thinks that this is identified

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as a ribose because of this 2 prime
hydroxyl?  Thank you.

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And who believes it's the 3 prime
hydroxyl that identified riboses?

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OK.  We have a smaller but firm
contingent.  In fact,

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it's the 2 prime hydroxyl that
identifies this is ribose.

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You remember, and you really need
to remember that this three prime

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hydroxyl is the reactive group that
allows the sugar phosphate backbone

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to polymerize.
This 2 prime hydroxyl is a reactive

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group.  It identifies this as ribose
rather than deoxyribose,

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and it also is an additional
reactive group.

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And the fact that it is a reactive
group makes RNA rather labile.

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OK?  So let's write a couple of
important things here.

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So this is RNA as the nucleic acid
polymer.

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You should really know this.
Ribose has both a 3 prime hydroxyl

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and this 2 prime hydroxyl.
And this is a reactive group.

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And because of this RNA is a much
less stable polymer than DNA.

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Here's another one.  What type of
polynucleotide is this and how do

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you know?  Yes.
You.  OK.  It's RNA.

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And it's RNA we know because of
these uracil groups.

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OK?  So uracil is an alternate base
to thymine that's found only in RNA.

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Here are the Us.  It tells you it's
RNA.  OK?  So you need to know those

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facts about RNA.

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Good.  So let me pose a question to
you.  In this litany that you've had

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several times now where the flow of
information moves from DNA to RNA to

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protein, why is the RNA there?
This is a rhetorical question.

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I'm going to try to answer it for
you.

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Why is the RNA there?
Why is there an RNA intermediate?

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You could imagine that the DNA

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double helix could open up and that
nucleic acid could be directly

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translated or could be directly
converted or the code could be

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changed to form a protein without
any RNA intermediate.

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But, in fact, universally throughout
biology, throughout our world anyway,

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throughout our earth,
RNA is there as an intermediate.

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Why?  Well, I think the answer
actually lies in evolution.

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RNA is probably the most ancient of
the information polymers.

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That is widely believed now.
So RNA is ancient.  It was the

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first, strongly believed now that it
was the first information

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carrying polymer.
RNAs themselves were catalytic.

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They became able to replicate.  And
they also probably became able to be

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translated into protein before DNA
was invented.  OK?

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So DNA's chemical structure is
different and it's a derivative of

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ribonucleic acid,
and undoubtedly came second.

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There was an advantage of having
DNA because it's so much more stable,

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and it made the hereditary material
much more stable and much more

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faithfully transmitted from
generation to generation.

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So RNA was ancient.
And the relationship between RNA

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and protein is probably a very old
one, and we'll talk about this

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relationship next lecture.
And I believe that that

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relationship has persisted,
and then DNA was kind of an add-on.

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And the DNA to RNA to protein does
not necessarily reflect the only way

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or the best way to do things.
Evolution is a capitalization of

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various changes.
And RNA to DNA, DNA to RNA to

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protein is how things work now.
But this, I think, is a consequence

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of the evolutionary past.
Now, however, in our modern world

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RNA serves two main purposes.
One of the things it does is to

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allow one to use just a subset of
the genes to make proteins.

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So, as you've been told several
times, you and I have about 30,

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00 genes in our genomes.  Not all of
those genes, and we will discuss

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this in great depth as the course
goes on.  Not all of those genes are

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used at any one time.
We use just a subset of the genes.

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And having them converted into an

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RNA intermediate is one of the ways
that you can allow just a subset of

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the genes to be used.
So I'm going to write here subset.

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Subset of gene usage.

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OK?  Because you can turn just some
of those genes,

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or you can convert some of those
genes into RNA,

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00:18:00,000 --> 00:18:04,000
the information in some of those
genes into RNA.

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And the other thing it lets you do
is to amplify the signal from each

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gene.  So there are two copies of
each gene in a diploid cell.

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When it comes to RNA there can be
up to 10,000 copies of RNA per cell

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of a particular RNA.
OK?  So you can get an

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amplification of the signal --

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-- from each gene.
RNA copy number per cell ranges

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from about one copy to about 10,
00, that's rare, copies per cell.

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All right.  So here we are.  Why
RNA? We've dealt with that.

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So I want to talk to you about two
things.  I want to talk to you about

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synthesizing the RNA,
and then I'm going to talk to you

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about modifying the RNA a bit.
And the first thing I want to cover

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is something called transcription.

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Which is also known as RNA synthesis.
And you all should have this

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handout.  So I'm not going to draw
it but I will write some salient

221
00:19:26,000 --> 00:19:35,000
features on the board for you.

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00:19:35,000 --> 00:19:42,000
And we're not quite ready to use
that.  I'm going to leave this up

223
00:19:42,000 --> 00:19:49,000
here, but I'm going to work on the
board for a little bit.

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00:19:49,000 --> 00:19:56,000
The basic idea behind transcription,
RNA synthesis,

225
00:19:56,000 --> 00:20:04,000
is that one copies a DNA template
into a complementary RNA strand,

226
00:20:04,000 --> 00:20:09,000
complementary RNA.
And one does this,

227
00:20:09,000 --> 00:20:13,000
as I've alluded to,
only from the genes.

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00:20:13,000 --> 00:20:24,000
And this is an interesting point

229
00:20:24,000 --> 00:20:31,000
because although you have 30,
00 genes in your genome, in fact,

230
00:20:31,000 --> 00:20:37,000
those 30,000 genes only take up
about 5% of the total amount of DNA

231
00:20:37,000 --> 00:20:44,000
in each of your cells.
So 5% of your total DNA of your

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00:20:44,000 --> 00:20:51,000
genome comprises the genes,
the information carrying entities in

233
00:20:51,000 --> 00:20:58,000
your DNA.  And the rest
is other stuff.

234
00:20:58,000 --> 00:21:03,000
So the 95% is not genes.
It consists of various repeats,

235
00:21:03,000 --> 00:21:09,000
repetitive DNA that can be there at
just a few copies per genome or at

236
00:21:09,000 --> 00:21:14,000
10,000 copies per genome.
They can be real little, 10 base

237
00:21:14,000 --> 00:21:20,000
pairs, six base pair repeats,
or they can be a few kilo bases

238
00:21:20,000 --> 00:21:25,000
repeated many times.
Oris, Origins of Replication that

239
00:21:25,000 --> 00:21:31,000
you talked about last
time are not genes.

240
00:21:31,000 --> 00:21:36,000
Those are there,
too.  Centromeres,

241
00:21:36,000 --> 00:21:42,000
the middles of chromosomes.
Telomeres, the ends of chromosomes.

242
00:21:42,000 --> 00:21:47,000
All of these things are not genes,
and they comprise the bulk of your

243
00:21:47,000 --> 00:21:53,000
DNA.  Now, this isn't true in all
organisms.  OK?

244
00:21:53,000 --> 00:21:59,000
Some organisms have got very little
of this repetitive extra DNA.

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00:21:59,000 --> 00:22:06,000
We happen to have a great deal of it.
OK.  So let's pursue this a bit

246
00:22:06,000 --> 00:22:14,000
more.  And let's think a bit more
about these genes.

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00:22:14,000 --> 00:22:22,000
And in particular let's think about
the kinds of RNAs that those genes

248
00:22:22,000 --> 00:22:30,000
make.  So I'm going to talk about
gene classes or classes.

249
00:22:30,000 --> 00:22:37,000
And this is with respect to the RNA
and the functional RNA that comes

250
00:22:37,000 --> 00:22:44,000
from those sets of genes.
And I want to distinguish two major

251
00:22:44,000 --> 00:22:52,000
classes of genes.
The first are the protein encoding

252
00:22:52,000 --> 00:22:59,000
genes.  And protein encoding genes
move through a type of RNA that is

253
00:22:59,000 --> 00:23:07,000
called messenger RNA,
abbreviated mRNA.

254
00:23:07,000 --> 00:23:16,000
Messenger RNAs comprise about 1% of
the total amount of RNA in a cell.

255
00:23:16,000 --> 00:23:25,000
And they can range in size from
let's say 100 base pairs to 10,

256
00:23:25,000 --> 00:23:32,000
00 base pairs.  OK?
So there's a very wide size range.

257
00:23:32,000 --> 00:23:38,000
No, not base pairs.  Yell at me.
Why not base pairs?

258
00:23:38,000 --> 00:23:43,000
Why was I wrong saying base pairs?
Tell me about RNA.  Raise your hand.

259
00:23:43,000 --> 00:23:49,000
This is worth a frog.
I caught myself, but if you can

260
00:23:49,000 --> 00:23:54,000
catch me, too.
Yes.  You.  Good.

261
00:23:54,000 --> 00:24:00,000
OK.  Generally RNA is single,
woops.

262
00:24:00,000 --> 00:24:04,000
RNA is single-stranded.
It does not form, it can form a

263
00:24:04,000 --> 00:24:08,000
double helix, OK?
It's not as stable as the DNA

264
00:24:08,000 --> 00:24:12,000
double helix, and many RNAs,
probably most RNAs have some

265
00:24:12,000 --> 00:24:16,000
double-strandedness to them,
but that is an intromolecular double

266
00:24:16,000 --> 00:24:20,000
strand in this.
There are some RNAs that form

267
00:24:20,000 --> 00:24:24,000
intermolecular double strands,
but in generally I'm going to assume

268
00:24:24,000 --> 00:24:28,000
that RNAs are single-stranded.
So we talk about 100 bases rather

269
00:24:28,000 --> 00:24:33,000
than 100 base pairs.
OK?  Second class of genes are the

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00:24:33,000 --> 00:24:39,000
ones that do not code for protein,
and in this case the RNA is the

271
00:24:39,000 --> 00:24:45,000
final product.
And this litany of DNA to RNA to

272
00:24:45,000 --> 00:24:51,000
protein doesn't hold.
You just stop at the RNA.

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00:24:51,000 --> 00:24:58,000
And the RNA is the functional thing.
So here RNA is the final product.

274
00:24:58,000 --> 00:25:06,000
And we can break these into a bunch

275
00:25:06,000 --> 00:25:12,000
of different classes.
Ribosomal RNAs, abbreviated rRNA

276
00:25:12,000 --> 00:25:19,000
are a very abundant class of RNA
that comprise about,

277
00:25:19,000 --> 00:25:25,000
I've moved over here,
let me move here, 98% of total RNA.

278
00:25:25,000 --> 00:25:31,000
And there are a few thousand bases
in length that say 2,

279
00:25:31,000 --> 00:25:37,000
00 to 4,000 bases in length.
OK?  So this is 98% ribosomal RNA.

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00:25:37,000 --> 00:25:42,000
This is fascinating.  I'll tell you
next time.  This is the RNA that

281
00:25:42,000 --> 00:25:47,000
comprises a very large proportion of
the ribosome that is the factory

282
00:25:47,000 --> 00:25:52,000
that makes the proteins.
OK?  And so I will tell you more

283
00:25:52,000 --> 00:25:57,000
about these next time.
Some other ones, tRNA,

284
00:25:57,000 --> 00:26:03,000
the T for transfer RNA.
tRNA comprise about 1% of all RNA

285
00:26:03,000 --> 00:26:11,000
and are about 100 base pairs,
100 bases long.  OK? And then an

286
00:26:11,000 --> 00:26:19,000
interesting one that MIT has had a
huge role in discovering and

287
00:26:19,000 --> 00:26:27,000
studying, these things called micro
RNAs, abbreviated miRNAs,

288
00:26:27,000 --> 00:26:35,000
which are, they're at relatively low
abundance.

289
00:26:35,000 --> 00:26:43,000
Less than 1% of total RNAs.
And these are small.  In their

290
00:26:43,000 --> 00:26:51,000
mature form they're about 22 bases
in length.  OK.

291
00:26:51,000 --> 00:27:00,000
So now, and I believe I cannot do
anything with these boards.

292
00:27:00,000 --> 00:27:03,000
Ah, I can do something with this one,
but that one is stuck.

293
00:27:03,000 --> 00:27:07,000
All right.  So I'm going to do
something with this one.

294
00:27:07,000 --> 00:27:10,000
And then I'm afraid it's going to
disappear, but it's not going to

295
00:27:10,000 --> 00:27:14,000
matter because you have the handout
in front of you.

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00:27:14,000 --> 00:27:18,000
So now I'm ready to move on with
you to the basic idea of

297
00:27:18,000 --> 00:27:21,000
transcription.
And I'm going to write some facts

298
00:27:21,000 --> 00:27:25,000
on the board, and we're going to
look at these cartoons that I drew

299
00:27:25,000 --> 00:27:29,000
for you together because I think
your book is kind of difficult.

300
00:27:29,000 --> 00:27:36,000
So I decided to draw some cartoons
to help you with the basic idea.

301
00:27:36,000 --> 00:27:44,000
Transcription or RNA synthesis
takes place in the nucleus.

302
00:27:44,000 --> 00:27:59,000
Anyone else need a handout?

303
00:27:59,000 --> 00:28:05,000
Why don't you come on down.
Actually, one of the TAs, could you

304
00:28:05,000 --> 00:28:11,000
be an emissary and just hand out to
those people with raised hands?

305
00:28:11,000 --> 00:28:18,000
Thanks.  Transcription takes place
in the nucleus.

306
00:28:18,000 --> 00:28:24,000
And the idea is really analogous to
DNA replication with a difference.

307
00:28:24,000 --> 00:28:30,000
The analogy is the synthesis of a
complementary strand of nucleic acid

308
00:28:30,000 --> 00:28:37,000
on a template strand.
So this is an enormously important

309
00:28:37,000 --> 00:28:43,000
principle that you need to have.
Super important that you get the

310
00:28:43,000 --> 00:28:50,000
principle.  The basic idea involves
synthesis of a complementary strand

311
00:28:50,000 --> 00:28:57,000
of nucleic acid from a template
strand.  The template,

312
00:28:57,000 --> 00:29:04,000
actually, let me start even earlier
than that.

313
00:29:04,000 --> 00:29:09,000
We start with a gene that generally
comprises double-stranded DNA.

314
00:29:09,000 --> 00:29:14,000
There are exceptions to almost
everything that I will tell you,

315
00:29:14,000 --> 00:29:19,000
or that Professor Jacks will tell
you.  You should understand that

316
00:29:19,000 --> 00:29:25,000
there are exceptions.
Some organisms, particularly

317
00:29:25,000 --> 00:29:30,000
viruses have genomes that are RNA
that can be single-stranded or

318
00:29:30,000 --> 00:29:35,000
double-stranded RNA.
Some have genomes that are

319
00:29:35,000 --> 00:29:40,000
single-stranded DNA.
But in general most genomes are

320
00:29:40,000 --> 00:29:45,000
double-stranded DNA.
And the deal is this.

321
00:29:45,000 --> 00:29:50,000
The double-stranded DNA separates
its strands, and one of the strands,

322
00:29:50,000 --> 00:29:55,000
and this is the difference between
DNA replication and transcription,

323
00:29:55,000 --> 00:30:01,000
one of the strands becomes the
template strand.

324
00:30:01,000 --> 00:30:14,000
And this template is copied to form

325
00:30:14,000 --> 00:30:22,000
a complementary strand.
And it's copied by an enzyme called

326
00:30:22,000 --> 00:30:31,000
RNA polymerase.
So RNA polymerase synthesizes the

327
00:30:31,000 --> 00:30:40,000
complementary strand to
the template strand.

328
00:30:40,000 --> 00:30:45,000
complementary strand.
And it does so, of course,

329
00:30:45,000 --> 00:30:51,000
as RNA, because we're talking about
RNA synthesis and this is RNA

330
00:30:51,000 --> 00:30:57,000
polymerase.  It does not,
unlike DNA polymerization,

331
00:30:57,000 --> 00:31:03,000
require a primer.  So this does not
require a primer.

332
00:31:03,000 --> 00:31:07,000
OK.  You should know,
and it should be getting deep within

333
00:31:07,000 --> 00:31:11,000
your neural circuitry that
polymerization occurs by adding

334
00:31:11,000 --> 00:31:15,000
nucleotides to the 3 prime end of
the growing polymer.

335
00:31:15,000 --> 00:31:20,000
Yes.  If that didn't make,
you know, if you didn't say "yeah"

336
00:31:20,000 --> 00:31:24,000
to that, go back and think about it,
go back and look at problem sets and

337
00:31:24,000 --> 00:31:28,000
you'll get more practice in this.
But you really need to know that

338
00:31:28,000 --> 00:31:33,000
the growing chain adds
onto the 3 prime end.

339
00:31:33,000 --> 00:31:39,000
OK.  So after the polymer,
after the RNA polymer is made the

340
00:31:39,000 --> 00:31:46,000
RNA is released from the template
strand.  As its being transcribed it

341
00:31:46,000 --> 00:31:52,000
forms this complementary strand.
And, as you know, complementary

342
00:31:52,000 --> 00:31:59,000
strands can base pair.
After it's made it is released from

343
00:31:59,000 --> 00:32:05,000
the template.
And it usually then goes into the

344
00:32:05,000 --> 00:32:09,000
cytoplasm where it does its thing.
So if you look at the diagram I

345
00:32:09,000 --> 00:32:13,000
gave you, that's what's up here,
here's your double-stranded DNA,

346
00:32:13,000 --> 00:32:17,000
your gene.  The strands separate.
One strand is transcribed into RNA.

347
00:32:17,000 --> 00:32:21,000
The RNA is release.  Obviously,
your double-stranded template,

348
00:32:21,000 --> 00:32:25,000
or what was your double-stranded
template will reform

349
00:32:25,000 --> 00:32:30,000
its double strand.
So perhaps that's not so obvious,

350
00:32:30,000 --> 00:32:36,000
but the double-stranded, originally
double-stranded template will reform

351
00:32:36,000 --> 00:32:41,000
its double strands,
thus released RNA, then goes into

352
00:32:41,000 --> 00:32:47,000
the cytoplasm where it is translated
into protein, or where the RNA is

353
00:32:47,000 --> 00:32:53,000
the final product.
So let's look at that in a bit more

354
00:32:53,000 --> 00:32:58,000
detail.
I've got here a template strand

355
00:32:58,000 --> 00:33:03,000
shown in red.  This is,
again, the second picture in the

356
00:33:03,000 --> 00:33:09,000
handout in front of you.
And I've got three features added

357
00:33:09,000 --> 00:33:14,000
here.  I have got a precise start
site of transcription.

358
00:33:14,000 --> 00:33:19,000
I've indicated elongation where the
polymer is elongating.

359
00:33:19,000 --> 00:33:24,000
And I have a precise termination
site where transcription ends.

360
00:33:24,000 --> 00:33:30,000
OK?  Now, let me see what I have
here.

361
00:33:30,000 --> 00:33:35,000
I have a movie here.
Watch the movie.  I'll show it to

362
00:33:35,000 --> 00:33:41,000
you once, and then you can go and
watch it at your leisure.

363
00:33:41,000 --> 00:33:47,000
This is meant to be RNA polymerase.
There's the helix opening up

364
00:33:47,000 --> 00:33:53,000
locally.  Here are ribonucleotide
triphosphates coming in,

365
00:33:53,000 --> 00:33:59,000
and RNA polymerase is catalyzing
their synthesis.  OK?

366
00:33:59,000 --> 00:34:03,000
So the template strand is the bottom
and here is the RNA being released.

367
00:34:03,000 --> 00:34:08,000
There's RNA polymerase moving along
the helix.  And the depiction is

368
00:34:08,000 --> 00:34:13,000
that the helix is opening locally
and then closing again behind the

369
00:34:13,000 --> 00:34:18,000
RNA polymerase.
At transcription termination,

370
00:34:18,000 --> 00:34:23,000
the helix, the gene helix zips up
again and the transcript is released.

371
00:34:23,000 --> 00:34:28,000
So this is a very much
simplified story.

372
00:34:28,000 --> 00:34:32,000
But is the basic principle of
transcription.

373
00:34:32,000 --> 00:34:37,000
And you should know it.
And in particular I have put onto

374
00:34:37,000 --> 00:34:41,000
this second diagram,
and because you have him in front of

375
00:34:41,000 --> 00:34:46,000
you I'm not going to write this on
the board, I'm going to use this as

376
00:34:46,000 --> 00:34:50,000
something to tell you,
I have put the directionality of the

377
00:34:50,000 --> 00:34:55,000
strands of the double-helix on this
diagram.  This should be something

378
00:34:55,000 --> 00:35:00,000
you can deal with.
5 prime to 3 prime on one strand.

379
00:35:00,000 --> 00:35:04,000
The other strand is anti-parallel.
RNA, any nucleic acid is

380
00:35:04,000 --> 00:35:08,000
synthesized by adding onto the 3
prime end.  And that newly

381
00:35:08,000 --> 00:35:12,000
synthesizing nucleic acid polymer is
anti-parallel to the template.

382
00:35:12,000 --> 00:35:16,000
This is also something that you
should be familiar with.

383
00:35:16,000 --> 00:35:20,000
And you will have, will have,
have not yet, will have practice on

384
00:35:20,000 --> 00:35:24,000
doing this kind of polymerization,
but it should be something you

385
00:35:24,000 --> 00:35:28,000
really, really should be familiar
with, this anti-parallel

386
00:35:28,000 --> 00:35:32,000
requirement.
So, in fact, you can tell the

387
00:35:32,000 --> 00:35:36,000
direction of transcription because
of the directionality of the

388
00:35:36,000 --> 00:35:40,000
template strand.
OK.  So this is very important for

389
00:35:40,000 --> 00:35:45,000
you to go and think about after
class the directionality of the

390
00:35:45,000 --> 00:35:49,000
template and of the newly
synthesized polymer.

391
00:35:49,000 --> 00:35:54,000
These are some diagrams from your
book, and you can go and look at

392
00:35:54,000 --> 00:35:58,000
them.  I'm not going to dwell on
them.  They indicate the different

393
00:35:58,000 --> 00:36:02,000
between, or the steps in
transcription initiation,

394
00:36:02,000 --> 00:36:06,000
elongation and termination.
And I've put them up there just to

395
00:36:06,000 --> 00:36:10,000
tell you there are these diagrams in
your book and you can go and take a

396
00:36:10,000 --> 00:36:14,000
look at them and read the
accompanying text.

397
00:36:14,000 --> 00:36:18,000
OK.  So I see three problems with
transcription that are very

398
00:36:18,000 --> 00:36:28,000
interesting problems.

399
00:36:28,000 --> 00:36:33,000
One is how to find the genes.
I'll write them on the board and

400
00:36:33,000 --> 00:36:43,000
then we'll go through them.

401
00:36:43,000 --> 00:36:47,000
5% of the genome is genes.
That's most of it that is not genes.

402
00:36:47,000 --> 00:36:51,000
How does the transcription enzyme,
how does the RNA polymerase know

403
00:36:51,000 --> 00:36:56,000
which is a gene and which is not a
gene?  How does it know,

404
00:36:56,000 --> 00:37:00,000
even if it finds the gene,
which strand is the template strand

405
00:37:00,000 --> 00:37:05,000
and which is not the
template strand?

406
00:37:05,000 --> 00:37:08,000
I could have drawn your previous
diagram where the top strand was the

407
00:37:08,000 --> 00:37:11,000
template, and that what would have
happened would be that the RNA

408
00:37:11,000 --> 00:37:15,000
synthesis went in the other
direction.  So which strand

409
00:37:15,000 --> 00:37:31,000
is the template?

410
00:37:31,000 --> 00:37:38,000
And that also gives you the
direction, of course,

411
00:37:38,000 --> 00:37:46,000
of transcription.  And the third one
I'm going to write,

412
00:37:46,000 --> 00:37:54,000
and then I'll tell you about this in
a moment.  I'm going to write how to

413
00:37:54,000 --> 00:38:00,000
unwrap chromatin.  OK.
So in each of your cells,

414
00:38:00,000 --> 00:38:05,000
you have to look at me for this.
In each of your cells there is this

415
00:38:05,000 --> 00:38:11,000
length of DNA.
One meter.  This is a little over,

416
00:38:11,000 --> 00:38:16,000
but one meter of DNA.  How big is
the average cell in diameter?

417
00:38:16,000 --> 00:38:21,000
Give it to me in micrometers.
Worth a frog.  On average.  Well,

418
00:38:21,000 --> 00:38:27,000
that's actually a really big cell.
It's about ten times

419
00:38:27,000 --> 00:38:32,000
less than that.
But whoever that was,

420
00:38:32,000 --> 00:38:36,000
who was it?  No way.  These are very
bad to throw.  Very bad to throw.

421
00:38:36,000 --> 00:38:41,000
You can have it because you caught
it.  See me afterwards.

422
00:38:41,000 --> 00:38:46,000
I'll give you one.  OK.
[LAUGHTER]  OK.  So how do you pack

423
00:38:46,000 --> 00:38:50,000
a meter of DNA into a cell that is
about ten microns in diameter?

424
00:38:50,000 --> 00:38:55,000
OK.  So, OK, Jamie, you want to
hazard an answer here?

425
00:38:55,000 --> 00:39:06,000
Your hand was up.

426
00:39:06,000 --> 00:39:10,000
OK.  Good.  You can wind it up.
OK.  The other thing you have to do,

427
00:39:10,000 --> 00:39:14,000
of course, is to make it really thin.
It has to be a lot thinner than my

428
00:39:14,000 --> 00:39:18,000
piece of rope.
But once you've made it really thin

429
00:39:18,000 --> 00:39:22,000
you can then wind it up.
OK.  It's logical and this is how

430
00:39:22,000 --> 00:39:26,000
it's done.  And you can wind it up
and then it will fit into

431
00:39:26,000 --> 00:39:31,000
your ten micron cell.
Now, in actual fact,

432
00:39:31,000 --> 00:39:36,000
there's a whole process to do that.
And I'm going to go through them as

433
00:39:36,000 --> 00:39:41,000
we go through these problems here.
So here is problem one exemplified.

434
00:39:41,000 --> 00:39:46,000
I've got red little dots for each
of the genes.  How does RNA

435
00:39:46,000 --> 00:39:51,000
polymerase find these genes in this
vast amount of DNA that is not genes?

436
00:39:51,000 --> 00:39:56,000
Here's the other one.
Which strand is the template?

437
00:39:56,000 --> 00:40:01,000
Oh.
And here is a nice problem that in

438
00:40:01,000 --> 00:40:05,000
the interest of time I am not going
to do here in class with you,

439
00:40:05,000 --> 00:40:10,000
but I want you guys to go and do
this as an exercise.

440
00:40:10,000 --> 00:40:14,000
I will tell you that the answer is
not on your handout on the Web.

441
00:40:14,000 --> 00:40:18,000
I took it off.  Sneaky, ha?  So
that you can go and think about this.

442
00:40:18,000 --> 00:40:23,000
I want you to go and understand
that the products of synthesis from

443
00:40:23,000 --> 00:40:27,000
either strand of a DNA
double-stranded helix

444
00:40:27,000 --> 00:40:32,000
are not the same.
OK?  And I'm going to zip through

445
00:40:32,000 --> 00:40:37,000
this because I want to move on here.
OK.  And I want to move to problem

446
00:40:37,000 --> 00:40:42,000
three which is this thing I called
chromatin.  DNA is wound up around

447
00:40:42,000 --> 00:40:46,000
proteins.  These are called histones,
and we'll have more to say about

448
00:40:46,000 --> 00:40:51,000
them later in the course.
And wound up and wound up and wound

449
00:40:51,000 --> 00:40:56,000
up.  And there is a very set number
and type of proteins that the DNA is

450
00:40:56,000 --> 00:41:01,000
wound around.
And once the DNA has been wound

451
00:41:01,000 --> 00:41:05,000
around once, those DNA protein
complexes are wound up some more,

452
00:41:05,000 --> 00:41:10,000
and then wound up some more.  And
eventually you get them wound up and

453
00:41:10,000 --> 00:41:14,000
wrapped up so much you get the
characteristic rather large

454
00:41:14,000 --> 00:41:19,000
chromosomes which are very much
packed DNA.  Now,

455
00:41:19,000 --> 00:41:23,000
this is a great way to fit DNA into
a cell.  However,

456
00:41:23,000 --> 00:41:28,000
this wrapping up of the chromatin
into, the wrapping up of the DNA

457
00:41:28,000 --> 00:41:33,000
into this chromatin structure
inhibits transcription.

458
00:41:33,000 --> 00:41:37,000
And in order to allow transcription
to proceed, you have to remove these

459
00:41:37,000 --> 00:41:42,000
proteins from the DNA and allow it
to unwind locally.

460
00:41:42,000 --> 00:41:47,000
And that takes a whole series of
enzymatic steps,

461
00:41:47,000 --> 00:41:51,000
again that we'll explore more later
in the course.

462
00:41:51,000 --> 00:41:56,000
But the problem I throw out at you
now is hw do you unwrap the

463
00:41:56,000 --> 00:42:01,000
chromatin where transcription is
needed?  And the answer to all of

464
00:42:01,000 --> 00:42:06,000
these things lies in a
specific, no, stop.

465
00:42:06,000 --> 00:42:11,000
Stop. Down.  Up.
OK.  The answer to all of these

466
00:42:11,000 --> 00:42:17,000
questions lies in a specific DNA
sequence or a series of specific DNA

467
00:42:17,000 --> 00:42:23,000
sequences that are collectively
called --

468
00:42:23,000 --> 00:42:34,000
-- the promoter.

469
00:42:34,000 --> 00:42:41,000
Here's another one.
What is a promoter?  And I need to

470
00:42:41,000 --> 00:42:49,000
make the distinction now between
transcribed DNA of a gene and

471
00:42:49,000 --> 00:42:56,000
untranscribed DNA of a gene.
The promoter is part of a gene but

472
00:42:56,000 --> 00:43:03,000
it is not transcribed.
It usually depends on the gene and

473
00:43:03,000 --> 00:43:09,000
the type of gene.
It usually lies 5 prime to the

474
00:43:09,000 --> 00:43:16,000
transcriptional start site.
And it is a DNA sequence that says

475
00:43:16,000 --> 00:43:23,000
this is a gene,
and it also says transcription

476
00:43:23,000 --> 00:43:30,000
should proceed in this
direction.  OK.

477
00:43:30,000 --> 00:43:34,000
And the way it does these things,
I'm going to each of the answers to

478
00:43:34,000 --> 00:43:39,000
each of the problems now,
is that it binds proteins that

479
00:43:39,000 --> 00:43:44,000
specifically recognize the sequence
of the promoter.

480
00:43:44,000 --> 00:43:48,000
So you talked about the DNA
replication origin and proteins that

481
00:43:48,000 --> 00:43:53,000
specifically recognize the
nucleotide sequence of the origin.

482
00:43:53,000 --> 00:43:58,000
This is analogous.  There are
proteins that recognize promoter

483
00:43:58,000 --> 00:44:03,000
sequences which are similar but not
identical from gene to gene.

484
00:44:03,000 --> 00:44:09,000
So it binds proteins.
And these are called transcription

485
00:44:09,000 --> 00:44:21,000
factors.

486
00:44:21,000 --> 00:44:25,000
And these transcription factors bind
in a DNA sequence specific way.

487
00:44:25,000 --> 00:44:30,000
OK.  It also binds RNA polymerase
which I'm going to abbreviate RNA

488
00:44:30,000 --> 00:44:37,000
polymerase, RNA pol.
OK?  And the answers to the three

489
00:44:37,000 --> 00:44:46,000
questions are that firstly the
protein-DNA interaction is sequence

490
00:44:46,000 --> 00:44:55,000
specific, sequence specific,
and so this allows you to actually

491
00:44:55,000 --> 00:45:02,000
find the genes.
Secondly, and this is cool and I'll

492
00:45:02,000 --> 00:45:07,000
show you a picture of this in a
moment, the proteins interact with a

493
00:45:07,000 --> 00:45:12,000
promoter DNA differently on
different strands of the helix so

494
00:45:12,000 --> 00:45:17,000
they bind asymmetrically.
They may bind more to one strand

495
00:45:17,000 --> 00:45:22,000
than to the other strand.
And this gives directionality to

496
00:45:22,000 --> 00:45:27,000
the transcription so the protein
binding is asymmetric

497
00:45:27,000 --> 00:45:36,000
or strand specific.

498
00:45:36,000 --> 00:45:41,000
Not for all of these proteins but
for a significant number.

499
00:45:41,000 --> 00:45:46,000
And that helps you decide which
strand you're going to use as the

500
00:45:46,000 --> 00:45:52,000
template.  And thirdly these
proteins have got associated with

501
00:45:52,000 --> 00:45:57,000
them activities that will unwrap the
chromatin, that will unwrap the DNA

502
00:45:57,000 --> 00:46:03,000
from its protein complexes and allow
it to be accessible to the

503
00:46:03,000 --> 00:46:16,000
transcription machinery.

504
00:46:16,000 --> 00:46:20,000
OK.  Let's zip so I can show you
this.  You can look at this on your

505
00:46:20,000 --> 00:46:25,000
slides.  Here are some pictures from
your book.  Really important in this,

506
00:46:25,000 --> 00:46:30,000
don't move.  Really important in
this is a protein called TF2D which

507
00:46:30,000 --> 00:46:35,000
recognizes a sequence called,
that goes T-A-T-A-A-A.

508
00:46:35,000 --> 00:46:40,000
This is called the TATA binding
protein.  And it's really important.

509
00:46:40,000 --> 00:46:45,000
And it's the one thing that, the
major thing that gives asymmetry to

510
00:46:45,000 --> 00:46:50,000
this transcription,
set of transcription factors on the

511
00:46:50,000 --> 00:46:55,000
promoter.  Once TF2D has bound to
the promoter, other proteins come

512
00:46:55,000 --> 00:47:01,000
along, including these various other
things called BFHG and so on.

513
00:47:01,000 --> 00:47:05,000
And here's RNA polymerase.
And you can see this complex

514
00:47:05,000 --> 00:47:10,000
positioned asymmetrically on the DNA.
And this complex you should know

515
00:47:10,000 --> 00:47:15,000
the name of, I'm going to put it in
its own box here, is the

516
00:47:15,000 --> 00:47:23,000
initiation complex.

517
00:47:23,000 --> 00:47:28,000
And I want to show you a
crystallographic rendition of the

518
00:47:28,000 --> 00:47:34,000
TATA binding protein called TBP,
also sometimes called TF2D.  But

519
00:47:34,000 --> 00:47:40,000
TATA binding protein here shown in
purple.  And if you look here,

520
00:47:40,000 --> 00:47:45,000
you're looking head on at the double
helix.  OK?  Here's the helix.

521
00:47:45,000 --> 00:47:51,000
You're looking down the helix.
And you can see that this protein

522
00:47:51,000 --> 00:47:57,000
is positioned on just one side of
the helix, so that gives

523
00:47:57,000 --> 00:48:02,000
you asymmetry.
Here's another transcription factor

524
00:48:02,000 --> 00:48:06,000
bound to DNA.  This is a protein
called GAL-4.  It binds as a dimer.

525
00:48:06,000 --> 00:48:11,000
And you can see that GAL4 is the
blue.  And here it is contacting

526
00:48:11,000 --> 00:48:15,000
just one side,
one strand of this red double helix.

527
00:48:15,000 --> 00:48:20,000
And I'm going to stop there and
finish off the last little

528
00:48:20,000 --> 00:48:23,000
bit on Monday.