ERIC LANDER: So, now what I'd
like to do is turn to variations on the theme. One of the best ways to
understand what's going on with DNA goes to RNA goes to
protein is to consider how it works in different organisms. And the organisms we'll consider
are eukaryotes, like you; prokaryotes, like a
bacterium; and viruses. And each does the same basic
copying of nucleic acid to nucleic acid, transcription,
and translation. But there are some pretty
fascinating variations on the theme. So let's turn to those
variations. Let's start with DNA
replication. For eukaryotes, your genome are
long, double-stranded DNA molecules, and they're linear. Long, linear, double
stranded-- ds for double. I'll write it out-- double
stranded DNA. You know this. And you've got a lot of it. The human, you actually have
23 pairs of chromosomes. And the total length of your DNA
is about 3 times 10 to the ninth base pairs. So about 3 billion bases or so,
typical chromosome on the order of about 150 million
bases or so. The mouse, pretty similar. Mouse, 20 pairs of
chromosomes. And it's something like 2.7
times 10 to the ninth bases. The dog's about 2.5, the
elephant's about 3.3, essentially all mammals are
about 3 times 10 to the ninth. Tomatoes, 12 chromosomes. They're also in the
neighborhood of 3 times 10 to the ninth. You don't have more DNA than
a tomato, for example. Yeast, much smaller genome. Yeast is like-- it's got 16 chromosomes. Yeast is in the neighborhood
of about 13 million bases instead of billions of bases. Fruit flies, four chromosomes,
including a pretty measly one. And it's in the order of 200
million bases, et cetera. Many different sizes
for eukaryotes. But there's one significant
issue that all eukaryotes have, which is this chromosome
here, this linear chromosome, how do we replicate it? Well, we told you. We open up a bubble. We start making little
primers. Primase makes primers, you
extend it, it's all just fine. Except in one place, the end. Primase makes a primer,
continues to the end, no problem. What happens out there
at the end? Suppose primase sits
down here. What happens at the very
end of the chromosome? If my primer wasn't exactly at
the end of the chromosome, what happens? STUDENT: A little bit of it
doesn't get replicated. ERIC LANDER: A little bit
doesn't get replicated. Does that matter? Just a little bit-- STUDENT: [INAUDIBLE] ERIC LANDER: --per
cell division. Every cell division, you lose
a little bit of information off the end of your
chromosome. That's not so good . The word for end is tel. And so the ends of chromosomes
are called telomeres. And if you have a linear
chromosome, you have a problem with replicating your telomeres
because it's going to get a little short
each cell division. So what do you think
the cell does? Special mechanism, special
enzyme that comes along and adds-- there's a repeat sequence
that occurs. There's a repetitive sequence
that occurs out here. T2AGGG in humans, different
things in other organisms. And there's a specific enzyme
that comes along and adds back telomeres so you don't get in
trouble failing to replicate enough stuff at the end. By chance, the enzyme happens
to be called--? STUDENT: Telomerase. ERIC LANDER: Telomerase. And some folks got a Nobel Prize
for this last year, for understanding how telomerase
works. What cells in your body are
in need of replicating and replicating and replicating? Well, not necessarily
in your body. What cells in some people are
replicating and replicating and replicating and
replicating? Cancer cells. Cancer cells probably need
their telomerase, right? So one way to possibly treat
cancer might be to inhibit telomerase. So you see, all the things I'm
telling you about, these are useful fun facts to know and
tell about how the cell works. They're also the heart of a lot
of approaches in medicine. Because if you could
specifically inhibit telomerase, you might
specifically create a liability for rapidly
dividing cells. And so telomerase,
very interesting. Anyway, so a linear chromosome
has that problem. And if you understood our
mechanisms for replication, you'll understand there why the
linear chromosome needs some special mechanism. Now, here, prokaryotes
are much easier. Because most prokaryotes have
circular chromosomes, double-stranded circular-- ds circular-- DNA. This is much easier because
there are no ends. They don't have to worry about
the telomerase problem. They start somewhere, they start
replicating around, it all works fine. E. coli, for example, 4
million bases of DNA. The smallest are
microbacteria. They're on the order of about
a million bases of DNA. And they work just like
we talked about. But now, viruses,
they are weird. Viruses -- Turns out some viruses have some
double-stranded linear DNA, and some with multiple
chromosomes, even. Some viruses, though, have
double-stranded circular DNA. So it turns out viruses can
do either of those. It turns out they can
do more than that. Some viruses have
single-stranded circular DNA. That is to say, when they're
traveling around in their capsid, in the protein coat,
what's in the protein coat that you get infected with is
a single-strand of DNA. Well, how does it replicate? When it gets in, it becomes
double stranded. The first thing it has to do is
polymerases end up making this guy double stranded. But it travels around in its
single-stranded form. Why? Because it decided to. The great thing about viruses
is they are small. They've had a chance to
experiment with a zillion different things. But some viruses don't have
any DNA at all when they travel around. Instead of having DNA, they
alternatively could have RNA. Remember I said Crick already
figured out DNA and RNA are essentially equivalent. They're both nucleic acid. You can go from one
to the other. Some viruses decided to bring
along single-stranded RNA. So when the virus attaches to
the cell, it injects an RNA. The RNA is in the cell. But how is the virus going
to do anything? How's it going to replicate
itself? How do you replicate RNA? Well, the same stuff I told you
about replicating DNA-- namely, for replicating DNA
you use a DNA polymerase. It's a DNA-directed
DNA polymerase. It uses DNA as a template. Any reason not to have an
RNA-directed RNA polymerase? No. You can have one of those. So the way this works is
this gets replicated into a strand of RNA. It makes double-stranded RNA
by an RNA-directed RNA polymerase. That's a kind of weird enzyme. It takes RNA as its template. And it uses RNA as its
template, and it makes another copy. It makes a strand of RNA to
make it double stranded. And then it goes back and makes
another strand of RNA. But you don't have
that enzyme. Where does that enzyme
come from? STUDENT: The virus. ERIC LANDER: The virus? Did it bring it with it? STUDENT: Another cell
that it infects? ERIC LANDER: Another
cell that infects? STUDENT: The RNA encodes-- STUDENT: The RNA does-- ERIC LANDER: Whoa! Wouldn't it be cool if the RNA
was a messenger RNA and it encoded a protein, and the
protein it encoded was the RNA-directed RNA polymerase? Bingo. That is, in fact, what happens
with a certain class of what are called plus-strand
viruses. This is a messenger. It's a messenger RNA. And it's actually encodes the
instructions to the cell, please make me an RNA-directed
RNA polymerase. That's way cool. It also turns out that some
viruses are what are called minus-strand viruses. They don't bring a
messenger RNA. But instead, like you said,
they bring their own polymerase with them. The polymerase comes-- So here, these bring the
instructions for a polymerase. These actually bring the
polymerase itself. The polymerase then copies
this strand, which is the messenger RNA. And it makes more RNA-directed
RNA polymerases. So both of your two
solutions-- the virus brings a polymerase
with itself, or the virus brings the instructions
for the polymerase. Both of those actually happen. Pretty much a good rule with
viruses is anything that can happen does happen. This is pretty much Murphy's
rule for viruses there. Turns out viruses can
do one other thing. It turns out that viruses can
take that RNA strand-- RNA-- and, although I won't go into
all the details, copy that RNA strand into a DNA strand and
then copy that DNA strand to a second DNA strand, to make
double-stranded DNA. So some viruses that bring RNA
with them copy themselves not into more RNA, but
back into DNA. So instead of an RNA-directed
RNA polymerase or a DNA-directed DNA polymerase,
what is this? It's an RNA-directed
DNA polymerase. So this is an RNA-directed
DNA polymerase. In effect, what is
this thing doing? It's doing the exact opposite
of transcription. What's transcription? Reading DNA into RNA. What's this guy doing? Reading RNA into DNA. It's the reverse of
transcription. What is the enzyme called? Reverse transcriptase. It's called reverse
transcriptase. And then what happens is quite
insidious, is that if this is your own chromosomal DNA, that
piece of double-stranded DNA from the virus can be inserted
into your own human chromosome. The virus can then make more
copies of itself by transcription of that. This is a truly insidious virus
because it doesn't just infect your cells and grow. It infects your cells, turns
into double-stranded DNA, and installs itself. And how do you get that DNA
from the virus out of your chromosome? You don't. You can't get it out. It's stuck there. These things, because they work
in this fashion of back from RNA into DNA, have
the name retroviruses. That's what these are,
retroviruses. And can anyone name a particular
retrovirus? STUDENT: HIV. ERIC LANDER: HIV. And that's how HIV works. It turns out that David
Baltimore won a Nobel Prize for the discovery of reverse
transcriptase. And again, the reason I tell you
about all these mechanisms is A, they're cool, and
they're about biology. And B, they're medically
important. Why is reverse transcriptase
not just cool but medically important? Because you could inhibit it. If you wanted to fight the HIV,
if you wanted to fight the AIDS virus, you could come
up with chemicals that inhibit reverse transcriptase. And of course, the cocktails
that are given to patients who have been infected by HIV that
now keep them alive very, very nicely include reverse
transcriptase inhibitors. It's a very important thing. If you understand the biology
of retroviruses, you understand the targets you can
use for drug development. And they have saved
billions of lives. So again, I say this is not
entirely unimportant stuff. This is kind of cool stuff, to
understand how this works. All right. Next up, transcription. Let's turn to transcription. Transcription is a little
bit easier. Transcription varies. Here, eukaryotes, prokaryotes,
and viruses. Let's see, let's start
now with prokaryotes. Prokaryotes, pretty simple. I have a chromosome, I have my
promoter, I make my messenger RNA, I'm done. Just like I taught you before. Transcription looks just
like I told you. But for eukaryotes, it's
a little weirder. Transcription, I have my RNA. I have my promoter. I make my RNA. And my RNA, it turns out, gets
processed in all sorts of interesting ways. The three ways in which
eukaryotic RNA are processed-- first, there is a modification
put onto the five-prime end. It is called-- well, it's basically a
backwards G. It's a G triphosphate that is put on
backwards, so it goes GPPP, right there. And this is called a cap. And it's important for message
recognition and stability. Eukaryotes put a funny little
chemical modification there. Eukaryotes also do something
where somewhere near the end, they cut the message, and they
stick on a bunch of A's as a tail at the end. And this is also important for
message recognition and message stability
and all that. And this is called
the poly(A) tail. Most of the names are
quite reasonable. The tail of lots of A's is
called the poly(A) tail. So eukaryotic messages have a
cap at the front, the poly(A) tail at the back. But the truly weird thing that
they have is, if this is my eukaryotic message here, some
chunks of the message are cut out and discarded entirely. They are spliced out. You might make a longer message,
and whole chunks are spliced out. This is called splicing. Splicing throws out sequences. And it could start with a long
mRNA and make it a short mRNA. Now, Phil Sharp, who is on the
faculty here at MIT, won a Nobel Prize some years ago for
his discovery, together with someone else, of the phenomenon
of splicing. So Phil is really cool. You should talk to Phil. This splicing involves leaving
some things in and excising other things. The things that are -- the things that go out
are called introns. The things that stay
in and are not excised are called exons. The nomenclature is
a little nuts. If it stays in, it's an exon. If it goes out, it's
an intron. As I told you, the phenomenon
was discovered by Phil Sharpe at MIT. The nomenclature was
proposed by Wally Gilbert at Harvard, [LAUGHTER] who's a good friend. I'm teasing Wally. I'm teasing Wally, but Wally
Is responsible for this nomenclature that confuses
generations of students. Why is this called an intron? Not because it stays in,
but because it's an intervening sequence. It's an Intervening sequence
when it is called an intron. And once something was called
an intron, the other thing became an exon. But I've got to say, for all
purposes, to me, in means in an ex means out. But it's exactly backward. I've now said that
a few times. That may help you remember that
it goes the other way. Introns are intervening
sequences, okay? So there are some pretty
impressive splicing events that go on. A typical gene, might
start off 30 kilobases, 30,000 bases. And it might get spliced down
to 3 kilobases, 3,000 bases. But for example, the Factor
VIII gene that encodes the factor that hemophiliacs lack,
it starts off with 200 kilobases, 200,000 bases. And all but 10,000
are cut out. So it starts off 200,000, you
throw out 190,000, you get down to 10. The winner, the Duchenne
muscular dystrophy gene, starts out at 2 million bases. And it gets cut down
to 16,000 bases. You make 2 million bases of RNA,
and you throw out almost the entirety of it and
retain only 16,000. What a waste. Why do this? Why break up your genes in
patches of exons separated by big intervening spaces and then
make a big RNA and splice it together? Why do something that dumb? Yeah? STUDENT: [INAUDIBLE]? ERIC LANDER: Sorry? STUDENT: Are they recycled? ERIC LANDER: The nucleotides,
you mean, in the RNA? The nucleotides are recycled. But remember, you spent the
trinucleotides there. So that was an energy
expenditure. Well, it turns out the
energy expenditure, big deal, who cares? But it turns out that this is
actually very interesting evolutionarily. I'll just tell you
for a second. Maybe you'll forget it. In a given organism,
it might be more efficient to not have introns. Well, actually, there's
one use for it. If I had introns, the cell could
do alternative splicing. It could take the same message
and splice it different ways in different cells. Your liver might splice
the message one way to make one protein. Your muscle might splice the
message a different way to make another protein. So you could actually
make multiple, different mature messages. That's cool, and that's used,
and most genes actually have alternative splice forms. They can be spliced up
in different ways. It's also cool evolutionarily. Because it turns out that if
your genes are broken up into patches like that, when random
breaks happen in your genome, and this bit of chromosome
attaches to that bit of chromosome, as happens
sometimes-- you get hit by some little
radiation, it breaks something, it puts
it together-- the gene you could have
a functioning gene. Because since your cell knows
how to take a long message and splice it together, the fact
that there was a break and a reunion, if it happens in
one of those intervening sequences, would give you
a new gene, and a new functional gene. And so, in fact, some people
think that this is one of the tricks evolution uses to
create diversity is by breaking up its information like
that into little patches of files that can then be
recombined with each other in different ways. So those are the ideas. In any case, it happens
a great deal. Now, viruses. Viruses it turns out, will
behave with regard to this like the organism in
which they live. Prokaryotic viruses will
behave like prokes. Eukaryotic viruses will
behave like eukes. Now, let's turn to
translation. How does translation
work between--? Well, now here, eukaryotes,
for the most part, are well behaved. A eukaryote makes an mRNA, it
goes to the ribosome, the mRNA makes your protein, just
like I taught you. But now, prokaryotes
are a little weird. Prokaryotes, you know, their
messages are fine. None of this funny cap business,
none of these poly(A) tails, none
of this splicing. But here's the weirdness
that prokes do. This mRNA, one mRNA
might encode multiple different proteins. Multiple proteins. So this is one protein. This, multiple proteins. So what happens is this RNA, the
ribosome gets on here at a certain site. Now remember I told you the
ribosome finds the first AUG? Well, it's a little
more complicated. There's a particular ribosome
binding site that ribosomes like to go to. It turns out that this
prokaryotic message has several of those ribosome
binding sites. Ribosomes sit down. They then scan for the first AUG
that they see after that point and start making
a protein. So I might have several
different proteins all being made by one messenger RNA, one
RNA making multiple proteins. Why would I do that? Wouldn't it be simpler, make
your head hurt less, to have one gene making one protein
instead of this one mRNA encoding multiple proteins? STUDENT: They're related
to one another. ERIC LANDER: They're related. Tell me about that. STUDENT: Well, like their
functions, if they had similar functions. ERIC LANDER: If they had similar
functions, it might be very efficient to have
regulatory controls that made one message. And then I get all the proteins
made together, rather than having separate regulatory controls for each message. Suppose I'm a prokaryote, and
I'm dividing very rapidly, and I care about having
a small genome. It might be very efficient
to do it this way. So what related genes
might you put together on the same message? STUDENT: Pathway. STUDENT: Same pathway. Maybe if you're in charge of the
committee, you would say, I would like to arrange the
genes encoding the multiple steps of a biochemical
pathway. And that's often what happens. That's exactly what happens. So you can get co-regulation
of genes, of genes in the same pathway. And such a thing, which is
called polycistronic message. Polycistronic is an unhelpful
name for it, but it's what they call it sometimes, or an
operon, a regulatory thing that makes many such things. Now, why do bacteria do it? To minimize DNA. They just want to use as little
space as possible. So once it's invented a
regulatory control that turns on this message when I want to
make arginine, well, just stick all the genes on
the same message. It's cheaper, simpler,
less DNA needed. Now, who really has trouble
with the amount of DNA they can have? Do you have trouble with-- Is replicating your DNA a rate
limiting step in your replication as an organism? No. What is the rate limiting
step in your organismal replication, your having
offspring? STUDENT: [INAUDIBLE]. ERIC LANDER: Sorry? STUDENT: When it comes
to an age that-- ERIC LANDER: It's graduating
from MIT, getting a job, things like that, right? And DNA replication is not rate limiting, actually, right? Getting a degree, all that,
that's rate limiting. But for bacteria, who are
reproducing not every 20 years but every 20 minutes,
DNA replication is a rate limiting step. It's important to their
replication. And they want compact genomes. You, you don't care about
a compact genome. It's simply not a big metabolic
cost to you. Viruses-- think about viruses. Their genomes are tiny. They're much tinier even
than prokaryotes. Typical viruses might have
a few thousand or tens of thousands of bases. Bacteria were millions
of bases. Viruses sometimes might
only have 10,000 letters of nucleic acid. They really have to use their
information very compactly. So you know what viruses
do sometimes? Not all, but some? Some viruses make
a messenger RNA. They have a ribosome binding
site right here. And they start translating
the message-- let's give it a sequence. ACUUGAGCAA, and we'll put
an AUG in front of that. They can start translating
here. Whoops, I'm just going
to fix that. But you know what? There's also an AUG over here. They could start
using that one. And you know what else? If they found another AUG
off frame, some of them use that too. Remember, the first AUG in
normal messages get used to set the phase of the codons. But in theory, I could be
reading those codons in a different reading frame, shifted
over by one or shifted over by two. Some viruses are so clever
that they encode three different proteins smack on top
of each other by reading the same nucleic
acid sequence. It shifted three
different ways. It makes your head hurt to
think, how could I make a functional protein? I've got to manage to get three
separate functional proteins built out of a single
nucleic acid sequence. One, it's easy. Just tell me the amino acids. Now this becomes an interesting
puzzle. Can I make a sequence where read
one out of frame, it also makes a protein I want. And another one where-- And some viruses have evolved
to do that, showing you how much they care about economizing
their DNA. All right. So what have we got? We have DNA replicates. We've seen how it replicates. Its mostly the same by nucleic
acid copying, but all these variations-- linears, circulars, single
strand, double strand, RNA, reverse transcriptase,
transcription, translation. Look over these examples. And what they should do is help
solidify how these things work and how they're used in
slightly different fashions. Next time.
