Ahern’s BB 350 at OSU – 21. Translation 3 & Biotechnology


>>I’ve seen more smiling faces
today than I’ve expected. That’s good. What are your thoughts? Whenever I ask thoughts,
usually you’re like this. [LAUGHTER] Have a non-verbal thought. Nope. Feedback? Perspectives? Was it too easy?>>[INAUDIBLE]>>It wasn’t too easy, so
I should make it harder next time? Yes, ma’am.>>[INAUDIBLE] >>Yeah. My long answer
questions like those will tend to be specific when
they ask for several things. Yeah, you’re right. They are. And part of the reason
for doing that is I find that, actually,
students feel a little more
comfortable if they know exactly what I’m asking them. But if I say tell me about
translation or transcription, then it’s like, oh, my god. What am I going to do? And then they start writing
and anything and everything, hoping they get
something with it. So that’s part of the
reason for doing that, but I understand
what you’re saying. Other thoughts? Yeah?>>[INAUDIBLE] >>So you like more short answer
questions worth fewer points. Is that what I understand?>>[INAUDIBLE] >>Yeah, I’ll tell
you– when I do that– and I appreciate the feedback. But when I do that,
what I run into is people start
running out of time. Because if I make more–
and I’ve tried this. If I try to do more than three
long answers of any sort. Everybody goes, oh, I
can’t get your exam done, and it’s like uh. But I understand what
you’re– I think personally, the more questions you
have worth fewer points, the more territory
you can cover. And I certainly
sympathize with that. But one of the
things in this class that’s different– I
teach BB450, 451, and 350. And on the BB450 class, they
take a somewhat similar format exam as you guys do. And on the first exam, I try to
get as many questions as I can, so that– for what
your said, and that’s where I run into trouble. And that class has 400
students, and they’ll come with oh, my god,
it’s just too much. And it’s like, OK. Cut back. Yeah?>>[INAUDIBLE] >>Yeah, that’s also
a good question and another consideration. One of the reasons
for making them worth more points, of course,
is that there’s, in theory, more in them. But I will take your suggestion
of more short answer questions on the next exam. I’ll do that. So how many got
complementary right?>>[INAUDIBLE]>>Nobody’s looked? You haven’t looked?>>[INAUDIBLE] It’s C-O-M-P-L-E-M-E-N-T-A-R-Y.>>[INAUDIBLE] Well, let’s move forward. We have some territory to cover. I plowed through a lot
of translation last time, and so I’ve gotten through
the general mechanism of translation. And if we look at
translation and compare prokaryotic and eukaryotic,
the overall process is basically the same. The names of the proteins
and all the various things in eukaryotic are different. We don’t care about that. It’s the process that
matters, and the process– if you understand
the process for one, you understand the general
process for the others. And I pointed out some of the
differences as I’ve gone along. I’ll point out a couple
more differences today, but they’re not as great
as you might think. One of the places where
they’re different enough is in the response
to certain chemicals. So as you can imagine,
proteins, of course, have very specific structures. And when we look
at a ribosome, we have dozens of proteins
that are in there. So it’s not totally surprising
that those proteins that are in ribosomes might
be targets for drugs, and they are. And there are enough
differences in the structure of the proteins in the
prokaryotic ribosome compared to the eukaryotic ribosome
that a drug can specifically be designed to knock
out the function of a prokaryotic
ribosome, but not affect a eukaryotic ribosome,
and that turns out to be a really powerful thing. So any time you how
a compound that only works on one class of
organisms, then you’ve got a very specific
kind of antibiotic. And so the– I’ve got a
couple of examples up here. But there are drugs that
specifically target proteins or processes in prokaryotic
translation that are different than eukaryotic. And if you think
about it, if you were to knock out
translation for an organism, you’re going to kill the
organism, because the organism has got to make proteins in
order to divide, to be alive, to do the things
that organisms do. One of those drugs that
targets a process in ribosomes is this compound
called puromycin. And you don’t need to
worry about the structure, but if you look at the
structure, what you’ll see is that it at least partly
resembles the end of a TRNA. And that end of a
TRNA can actually make it into a ribosome. If it makes it into the
ribosome, what will happen is the ribosome will accept it,
but then it can’t process it. It can’t do anything
with it, because it doesn’t have the right
structure as an actual TRNA molecule does. So what this
puromycin will do is it will actually get attached
onto the end of a growing polypeptide, but
then it’s stuck. And so the ribosome
can’t process, the ribosome can’t
go any further, and everything just stops. So you’re stopping
the process right in the middle of
making a protein, and that’s actually how
puromycin can kill cells. Other antibiotics that
target processes in ribosomes include chloramphenicol. Cloramphenicol is an antibiotic
that will specifically hit the ribosome
prokaryotic cells and knock out prokaryotic cells. Tetracycline will also do that. If we turn our
attention to eukaryotes, I’ve already talked about
a significant difference in the structure of
eukaryotic messenger RNAs compared to prokaryotic
messenger RNAs. So I’m going to remind you
that eukaryotic RNAs underwent a considerable amount
of modification after they were made. First, at the
five-prime end there’s a cap that’s put on to them. That’s that unusual
nucleotide structure that I told you about earlier. Second, at the
three-prim end there’s what’s called a poly-A
tail that’s put on, and that’s just a string
of dozens and dozens and dozens of A residues. I was a little hurried when I
talked about this last time, so I’m going to
mention it to you here now, because I think it’s
an important and interesting phenomenon. These two modifications that
happen to eukaryotic messenger RNAs actually have a
couple of functions. One of them, as we’ll
see in a minute, is that it helps the translation
process to proceed efficiently. But another important
consideration for these is that these modifications of
the ends of the messenger RNA help to stabilize
the messenger RNAs. What does that mean? Well, cells, if you think
about it, make RNAs, and cells also break down RNAs. They have enzymes in
them called RNases. R-N-A-S-E. And RNase is an
enzyme that you can think of for RNA like we talked
about a nuclease for DNA, or an exonuclease for DNA. These RNases are
abundant inside of cells, and they are there
because cells, once they’ve made
a messenger RNA, they don’t want that messenger
RNA being around forever. We’ve talked about how it would
be inefficient, for example, to be making the
proteins necessary for lactose metabolism
if lactose isn’t there. So that means that
not only does the cell want to control when it
synthesizes that messenger RNA, but it also wants to be able to
break down that messenger RNA so it’s not floating around and
being translated into protein when the cell doesn’t
want that protein. So cells are full of RNases. If you ever work with
RNA in a laboratory, you’ll find that RNases
are some of the worst things that you’ll
ever encounter, because RNases are everywhere. If you touch anything, your
hand is so full of RNases that it will degrade any RNA
you’re trying to work with. And not only that, but the
RNases are very stable. I talked about earlier
RNase in the term being a very stable protein
that we could heat up, and it would remain
nature– that is, it would come
back to being active. And so RNases are very
problematic that way. Well, one of the things that
helps to stabilize a messenger RNA in a eukaryotic cell
is the cap and the poly-A. The cap actually
prevents exonucleases from starting at the five-prime
end and chewing inwards. It physically prevents
that from happening. So the five-prime end of
eukaryotic messenger RNAs is protected by the cap. At the three-prime
end, one of the reasons that cells put hundreds
of A residues in is because the RNases that
start chewing in on a cell are exonucleases. They start at the
end and move inwards. And the longer that
track of A’s is, the longer it takes the
RNases to degrade that tail. They will degrade a few
bases, and they will fall off, and they will degrade,
and they will fall off. So the longer that tail is,
the longer that messenger RNA will be around. So both the cap and the
poly-A tail on a messenger RNA help the messenger RNA
to be stable for a longer period of time. We can imagine there might be
some messenger RNAs in a cell that the cell would want to keep
for a longer period of time. And some messenger RNAs
cell that it wouldn’t want to keep for a
longer period time, and it wouldn’t have as
long of a poly-A tail on it, and that turns out
to be the case. Well, so we’ve got a cap. We’ve got a poly-A tail. And another thing that I want
to say about this actually relates to translation. Sorry, got the wrong thing here. This depicts the
translation process that occurs in a eukaryotic cell. The translation process. What you see in green
is the messenger RNA, and the five-prime
end with the cap is right here where my
pointer is, where it says 7MG. The three-prime end goes
all the way up around here. What you see is an
initiation complex, and we talked about
initiation complexes in the initiation phase
of translation before. In eukaryotic cells,
they’re more complex, and that initiation
starts with two things. One is a protein that
binds to the cap, and the other is a protein
that binds to the tail. And I don’t even
care if you know the names of those proteins. That’s not what matters here. So you’ve got a protein that
binds to the poly-A tail. You’ve got a protein
that binds to the cap. And then you have
several proteins– and you notice
all the IFs there? Remember, IF stands
for Initiation Factor. You see various
initiation factors that are binding to each
other and between there to make this structure
a sort of a circle. And it turns out that in
eukaryotic translation, that circle is
thought to play a role in keeping the ribosome close
to the initiation codon. What does that mean? Well, it means that as
the ribosome starts here where the AUG would
be, it translates, it gets through there,
and it’s over here, and it hits the stop codon. It’s not very far away
from the AUG again. Whereas if that end is not held
close as it is in this case, then the ribosome
would have less of a chance of coming
back to the AUG and starting another
round of translation. So this circle is thought to
facilitate that efficiency of the translation process. Now I mentioned when I talked
about prokaryotic translation that prokaryotes have
a sequence called the Shine-Dalgarno
sequence, which is located a few bases ahead
of the initiation codon. And I said it played
a role of helping to position the messenger
RNA at the proper place in the ribosome so that the AUG
was in that P-site, remember? But I said that the
eukaryotic cells do not have a Shine-Dalgarno
sequence, and you might wonder how is it then that
the ribosome gets positioned properly on eukaryotic
messenger RNA. And I’m going to partly
answer that question for you, as best we know it. The partial answer
to that question is if we examine the
sequences of messenger RNAs in eukaryotes,
the first AUG that starts from the
five-prime end is usually the place where
translation starts, meaning it’s not the
second or the third of the fourth or the fifth one. In prokaryotic cells,
that’s not the case. Prokaryotic cells, they need
to have some signal that says this is the AUG to
start translation from, because the first
one won’t necessarily be where the translation
is supposed to start. So in this case, what
the ribosome can do is it can start assembling
anywhere in here, and then it can slide down until
it hits the first AUG and says, ah here’s where we start
putting in methionine, and we start the
translation process. A prokaryotic cell
can’t do that, because the first AUG isn’t
necessarily where translation is supposed to start. Questions about that? You guys are too worn
out from the exam. I can tell. So that’s the
eukaryotic process. Elongation in
eukaryotic continues very much like elongation
in prokaryotes. There is the bringing in
of the TRNAs, like you saw. There is the covering
up of that bond between the TRNA
and the amino acid by that EFTU-like protein
that carries in the TRNAs. And there is a
protein-like EFG that does the translocation step. And yes, GTP is the
energy of translation in eukaryotic cells, as well. So a lot of similarities
to the prokaryotic process. Same thing happens when
we get to termination. There are some minor
differences in termination, but overall, the process
is basically the same. A stop codon appears
in the A-site. A release factor comes in and
basically pulls everything apart, and translation
stops, and the peptide is released, just like we
saw in the prokaryotic cell. So that’s the
translation process that I want to talk about for
both prokaryotic and eukaryotic cells. I want to talk for a few
minutes about things that happen after a protein has been made. Things that happen after
a protein has been made. One of the– and actually,
what these modifications are are they’re called
post-translational modifications,
and you’ve already seen some post-translational
modifications. For example, converting a
zymogen into an active protein involved cutting a
peptide bond, and that was a post-translational
modification. You’ve also seen
adding a phosphate or taking phosphates
off of a protein, and those are also
post-translational modifications. Post-translational
modifications are very important for getting a
protein into its final form to be functional. And one of the best
examples of this is the protein that
we know as insulin. Insulin is what we
call a peptide hormone. So we’ll talk a little
bit more later about how insulin is a hormone. But it’s a peptide hormone
because it’s a protein. And the protein
is made originally as a fairly long chain or,
a relatively long chain of amino acids. And the initial
thing that’s made in the synthesis of insulin is
a protein called preproinsulin. Preproinsulin. And you can see that
preproinsulin looks like this, and it’s got some
sulfhydryls that are the side chains of cysteine amino acids. And those side chains can–
the polypeptide can fold, and those side chains can be
brought into close proximity such that they react and form
disulfide bonds, like you can see right here. That formation of
the disulfide bonds is absolutely critical, because
in the further processing of insulin, what we see is
that a couple of peptide bonds are broken. We see one broken here. We see one broken here. And when that happens,
this guy that started out as one long piece now is
really two short chains, and the only things
that hold them together are those disulfide bonds. Insulin in the prepro form
doesn’t have any function, but insulin in this form
over here on the right is essential for our body to
be able to handle glucose. It’s essential for our body
to be able to handle glucose. Glucose, as I will talk
about in a little bit, is something that is essentially
a poison to our body. It’s essentially
a poison– we’ll see why that’s the case–
and insulin helps the body to deal with the poisonous
effects of glucose. So insulin’s a very
important protein, and it’s made in a longer
form and then processed down into a smaller form
that you can see here. I’ve talked about
how it is that RNAs can get broken down by RNases. And I’ve talked about how
exonucleases can break down DNAs to some extent. Proteins also get broken
down inside of cells. And you might think cells–
they’re making things, and they turn around
and break them down. Why they do these things? And they do them because
damage over time can occur. In the case of DNA, we
saw replication damage, or we saw chemical
damage that could happen that meant that parts of the DNA
had to be removed and replaced. In the case of RNAs, we can
have either damage occurring or having an RNA
floating around that we don’t want floating around. And we can also imagine that
with proteins we could either have chemical damage
that occurs to a protein to destroy its function,
or we could have a protein that, hey, we don’t want that
function around right now. We’re going to break
that protein down. Well, cells have structures
within them whose job it is to break down proteins. And how do they know
which ones to break down? Well, it turns out that
there’s a flag that’s put onto proteins that are
destined to be broken down. There’s a flag that’s
put on to them– and this actually
shows one of the flags. The most common
flag that’s on there is a small peptide
called ubiquitin. Ubiquitin attachment
to a protein flags that protein
for destruction. And one of the things that
will destroy a protein is a structure
called a proteasome. P-R-O-T-E-A-S-O-M-E. A
proteasome is basically a complex that breaks down
proteins into smaller pieces. And those smaller pieces
can be further degraded into making something that is
useful for the amino acids that are in it. Let’s see. So that’s what proteasome does. Another interesting
consideration in proteins– and this is
not a post-translational modification, but
this is actually a very interesting and
very odd amino acid that is actually put into
proteins as they’re being made, in some cases. It’s an amino acid that we
refer to as the 21st amino acid. We always talk about how
there are 20 amino acids that are found in proteins, so
what’s a 21st amino acid mean? Well, it means that when
people have examined proteins over the years they
would occasionally come across this amino acid
that’s known as selenocysteine. It’s called selenocysteine
In because that Se right there is the atom
known as selenium. Selenocysteine looks
just like cysteine, except for it has a selenium
in place of the sulfur. This amino acid can actually
be attached to some TRNAs and can be incorporated into
a growing polypeptide chain. Now that means it’s
not post-translational modification. It’s a translation
that’s happening that puts this into a protein. The mechanism by
which that happens is beyond what we’re going
to talk about in this class, but you should know
that selenocysteine can be incorporated during
the translation process. And there are several
proteins in our bodies and in virtually every cell that
contained some selenocysteine. So where we talk about
there being 20 amino acids, there really are 21. And this one gets in by an
odd mechanism, as I said. I’m not going to
talk about it here, but there is a way to get this
translated into a protein. The last thing I
want to talk about is something I mentioned
briefly earlier– yes, question?>>[INAUDIBLE] >>Yeah, what does
selenocysteine do that’s different from
the other amino acids? Because it’s got that
selenium ion in there, we could imagine that the
chemistry would be a little bit different and would allow for
different kinds of reactions to be catalyzed or
catalyzed in different ways, and that’s actually
what it does. It’s not a very
satisfying answer, but that’s what the selenium
part of that actually does. And it’s actually for
that reason why selenium is an essential trace nutrient. You have to be very careful
with selenium, though, because selenium at
anything above trace amounts is poisonous, so you
don’t just go out and gobble a bunch of selenium. I always worry about that
when I tell people, oh, yeah, here’s this great nutrient. Everybody goes, oh wow. I’m going to have a
whole bunch of that. Not good. The last thing I mentioned
earlier in the term, and I want to mention
it here again, because it’s appropriate,
and that is a protein complex called a chaperone. And a chaperone is
important to help a protein to fold properly. It helps a protein
to fold properly. And what it provides
is a chamber within which that protein
can fold without interacting with other outside things. So a chaperone is
important to help a protein to fold properly. Not all proteins
need chaperones, but some proteins
do need chaperones in order to fold properly. You’ll see up here that
there’s a complex of this, and this complex has
something called HSP70. HSP stands for
Heat Shock Protein. And when cells get
heat shocked, they will usually induce the
synthesis of chaperones. What does heat do to proteins? It unfolds them. What do chaperones do? They help to properly fold them. So heat shock proteins
are important in helping– some of the heat
shock proteins– are important in helping other
proteins to fold properly. And the two that we’ll talk
about in facts and terms of name, these are found in
E. coli– the GroEL, GroES, and it’s called the
GroEL-GroES complex. And you can see the
GroES is basically a cap, whereas the GroEL
is the chamber within which that protein is inserted and
it’s allowed to properly fold. After it is folded properly, it
exits, and everything is done. That’s the last of what I want
to say here about translation. I want to turn our
attention now to the use of our knowledge of proteins
and DNA and RNA to make things. To make things. So we hear about the
term genetic engineering. And there’s a big ballot
measure in Corvallis right now about genetic
engineering– you guys are aware of that or not. And people get very uptight with
that term, genetic engineering, but in fact, genetic
engineering is something that has occurred
in nature since day one. We are getting the
tools that nature uses to do genetic engineering. That’s what we’re doing. And because of
these tools, we’re able to do some pretty
incredible things, and I’ll give you a
couple of examples. Some of the most
remarkable products, some of the most remarkable
medicines that are available are available today only because
we have genetic engineering. Prime example– hemophiliacs. People who have hemophilia are
lacking a protein or proteins that allow their blood
to clot properly. In the days before human
genetic engineering of proteins, the only way we could treat
people who had hemophilia was either to take thousands
of gallons of human blood and purify out tiny,
microscopic quantities of the clotting factor
that those people lacked and then inject
it back into them so they didn’t bleed to
death every time they cut their finger. Well, that was fine and dandy
until we had a blood supply that got contaminated with HIV. And there were
dozens or hundreds of people who were hemophiliacs
who died because the blood supply was contaminated
that their blood clotting factors came from. And this all came about
about the same time as the genetic engineering
of this happened. Because of the fact that
we could take and isolate from human cells the gene
that codes for the missing clotting factor or factors,
we could put those genes into bacteria, have bacteria
make those clotting factors. HIV was never present. Nothing else was very present. And not only could
we make tiny amounts, we could make tons of it. Cost went down, quality
went up, and people lived much normal lives
as a consequence of that. That’s one example. There are hundreds of
examples that exist today. Human growth hormone–
one good example. Another good example is one
that just escaped my mind. Oh, blood clotting– the
anti-clotting factors. So there’s a protein called
Tissue Plasminogen Activator– TPA– that dissolves clots
that has revolutionized the treatment of certain heart
attacks and stroke issues where blood clotting
was involved. You can actually inject
this into a person and have their clot be dissolved
almost instantaneously, which is really remarkable. So biotechnology has a lot
of practical applications. The practical
applications are– I want to talk a little bit about
how we do all of those things, and hopefully show
you in the process that we’re not
really playing god, as people like to describe that. We’re not playing god. Well, we’ve talked a little
bit about separation. And I’ll remind you,
what you’re looking at on the screen is something
that we’ve already talked about– gel electrophoresis. And specifically, this is
agarose gel electrophoresis. And specifically what
you see on the screen is the separation
of DNA fragments. The separation of DNA
fragments is a critical process to be able to analyze and to be
able to isolate specific DNAs that are of interest to us. How do we get fragments of DNA? Well, there’s a
variety of ways that we can get fragments of DNA, but
one of the most remarkable of these was something that was
discovered back in the 1970s, and that was that
there were enzymes that were found in
bacteria that would cut DNA at specific sequences. They would recognize
a specific sequence, they would bind to
it, and they would cut the DNA at that point. These enzymes are called
restriction enzymes. They’re also called
restriction endonucleases, but I’ll call them
restriction enzymes here. Restriction enzymes. And these restriction
enzymes are found throughout the
bacterial kingdom. You look in a bacterial
cell, in all likelihood, it’s going to have some kind
of a restriction enzyme. I want to tell you a little
bit about how they work, what they do, and how
they’re really useful to us for biotechnology purposes. Well, actually, I’ll
start with that. We can imagine that if we
take a human chromosome that’s several feet long, and we
want some part of it that’s a few microns in
size, we would like to have a way to precisely
cut and to obtain that fragment of DNA
that we’re interested in. Well, to do that, that means
we have to be able to cut DNA, and we have to be able
to cut DNA in places that we would desire. And so one of the
ways we do things is by cutting that DNA
with restriction enzymes. These restriction enzymes can
be purified from bacteria, mixed with the DNA. And when they’re
mixed with the DNA they will find their target
sequence, bind to it, and then make cuts like what
you’ll see on the screen. So here’s an example
of one restriction enzyme called EcoRI. It’s one of the most
well-known restriction enzymes in the world. It recognizes the
sequence GAATTC. So I’m only showing a part
of a long DNA strand there, and the middle of it is
this sequence GAATTC. If you look at
GAATTC, you’ll notice that if you read the top
strand from the five-prime to the three-prime
direction, it reads GAATTC. And if you go to the bottom
right of the bottom strand, you’ll see, reading left
to right from five-prime to three-prime, it
also reads GAATTC. It’s something that molecular
biologists mistakenly call a palindrome. It’s not a palindrome
technically, but it’s a molecular
biological palindrome. The top strand reads the
same as the bottom strand. Most restriction
enzymes recognize palindromic sequences. And that turns out to be useful,
because the enzyme has evolved the ability to
cut, in this case, between A G and an A
within that sequence. By recognizing a
palindromic sequence, it can cut between
this G an A, and it can cut between this G and
A. And when it does that, you make a double-stranded
cut in the DNA. This type of cut
that this has made is called a staggered
cut, meaning that it’s got some
bases overhanging, as you can see there. And that actually helps if we
decide to put the pieces back together. Put the pieces back
together like a puzzle– those pieces will
fit if we put them back together in the
proper way, and I’ll talk about that in a bit. Well, actually, you
can see it on here. So here’s the pieces. We see the T compare with A,
T compare with A, A compare T, A compare T. We put
those back together. In this case, we
started with this and we start with this one. It’s no surprise it’s
going to go back together. But what if I took a
different DNA that had also been cut with EcoRI that
had the same overhang, and I put it back
with this one here? That is, I had one DNA here
and a different DNA over here. This might be from
an E. coli cell. This might be from a dog cell. If I did that, and I put it
together with DNA ligase– and here’s where that DNA
ligase that you’ve learned about is important, because it
puts pieces back together. If I do that, I have just made
what we call a recombinant DNA. And it just simply means we’ve
recombined two things together that weren’t together to start. And that might seem, again,
there’s this playing god thing. And there’s all
this concern that’s out there about– I shouldn’t
say all this concern. Some people have it. A lot of people don’t
have the concern. But some people are very
concerned about the fact that you make recombinant DNAs. Again, nature has been making
recombinant DNAs for billions of years longer than
human beings have been on the face of the earth. So this process is happening
and has been happening long before humans were ever here. We’re using it to put things
together to make products. We can make human insulin. We can make human
growth hormone. We can make any
human protein that’s needed by using these
techniques that I’m describing. So restriction in the
nucleases are important. And I started to tell you
why they’re interesting, and I got distracted,
because I’m an old guy, and I get distracted. They’re important. They’re really interesting. Restriction of the
nucleases are in bacteria not just to give us
something to play with, but they actually play
a role in bacteria to help protect them
against viruses. How does a virus work? Well, we’re going to talk about
viruses, probably on Friday. But viruses work by
attaching to a cell. And viruses have a protein
coat on the outside that grabs ahold of a
cell, and once they’ve gotten ahold of
their target cell, they can inject nucleic
acid into the cell that codes for some proteins
that the virus wants. If nothing else happens,
then that virus will go in, it will replicate, and it most
likely will destroy the cell. Viruses are known for every
cell on the face of the earth. Bacteria get viruses
just like we get viruses. Well, in our case, we
have an immune system that’s protecting us
against the virus. We might catch a
cold, but we don’t die from a cold, because our
immune system catches up, destroys the virus, and
we get over the cold, and everything is
fine and dandy. A bacterial cell doesn’t
have an immune system, because a bacterial
cell is a single cell. How does a bacterium protect
itself against a virus? Well, the way that it does
it is with a restriction endonuclease. Imagine that that cell
is bound by a virus. The virus injects its
DNA into the cell. And the cell has a
restriction endonuclease that can cut the nucleic
acid of the virus. You would kill the
virus in the process. And that’s why bacteria
have restriction enzymes. They’re protection
against invading DNAs. Well, if you think
about that, you might ask the
question, well, how does it keep from
cutting its own DNA, because its own DNA is going to
have these sequences, as well. These sequences
are fairly common. This sequence here will occur
on average about once every 4000 nucleotides. How does the virus keep
from cutting up its own DNA? Virus– how do bacteria, sorry. How do bacteria keep from
cutting up its own DNA? The bacteria, in addition
to having restriction endonucleases, also have
something called a modification system. So the restriction endonuclease
is part of the system. The other part of the
system is another enzyme. So the restriction enzyme
recognizes the sequence and cuts it. The modification enzyme is
an enzyme called a methylase. M-E-T-H-Y-L-A-S-E. Every
time a bacterial cell has a restriction endonuclease,
it also has to have a corresponding methylase. What does the methylase do? The methylase recognizes
exactly the same sequence as the restriction enzyme. So the methylase
recognizes GAATTC. But instead of
cutting that sequence, what the methylase does
is it puts methyl groups on that sequence. It’s putting methyls onto
the bases of that sequence. So what? Who cares? Well, who cares is the cell,
because the restriction enzyme will not cut if that
sequence has been methylated. So the bacterium can
put methyl groups on that sequence in its own DNA. Its own DNA will not
get cut by the enzyme. But when this bacterial DNA
comes floating into the cell, this doesn’t have
methyl groups on it. Restriction enzyme cuts it. The virus is dead. You might say, well, what
if the methylase– can it put a methyl group
on the bacterial DNA? And the answer is, yes, it can. This always gives people
worry, because they want nature to be perfect. And nature is not perfect,
but nature is pretty good. Usually, the restriction
enzyme gets there first. Usually, the virus
gets broken down. But because viruses
exist, we know it doesn’t happen every time. Sometimes the methylase wins. If the methylase wins, then
the viral DNA is protected, and the virus is probably
going to kill the cell. Make sense? So restriction
modification systems are very important systems
for protecting bacteria. We don’t have restriction
enzymes in our cells. We don’t have them in our cells. Bacteria have them. We don’t have them, but we
also have an immune system, so we have a different
set up for protection then what bacteria do. There’s the methylation. There’s the modification. There’s target sequence,
protection, et cetera. This shows a variety
of restriction enzymes, and no, you don’t need to
memorize these sequences. But I show you that there
are different enzymes that recognize different sequences. Some of them recognize
staggered sequences, like EcoRI. There’s that GA and there’s that
GA, so that we have overhangs. BamHI cuts between G and
G and between G and G, and it leaves
staggered overhangs. Some only recognize for
base sequences, like HaeIII, and it cuts between G and C,
and it cuts between G and C. And if you look at
this one, you say, it doesn’t leave
a staggered cut. It leaves what we call a blunt
end, and that’s fine, too. There’s all kinds of
examples about how these cut. Here’s one called NotI that
recognizes an eight base sequence. And so there’s a
variety of things that different cells have. The naming of these comes
from the name of the bacteria that they come from. So EcoRI comes from
E. coli, actually, that’s found in your gut. Well, I’ve already
talked about a way to make a recombinant DNA. This figure, hopefully, brings
it to life a little bit. You can see how it is that
we have a DNA that we can put another piece of DNA into. And it also introduces a term
that I haven’t talked about, but that is a very important
term for us in biotechnology, and this term is something
called a plasmid. You’ve probably heard
the term before. I want to make sure you
know what a plasmid is. So a plasma is a
small, circular DNA that replicates in bacteria. It’s a small, circular DNA
that replicates in bacteria. The size of a bacterial DNA is
about anywhere from 1 million up to about 10
million base pairs. The size of a
plasmid is typically on the order of a few thousand. So it’s much, much smaller
than the bacterial DNA, but it can replicate, and it
can replicate in the same cell as the bacterial DNA. Well, plasmids turn out to
be really useful for us, because the larger a
DNA is, the harder it is to handle without breaking it. If we try to handle–
meaning manipulate in a test tube– a bacterial
DNA, it’ll break before we do anything to it. It’s just too fragile. Plasmids, because
they’re smaller, are not so easily torn. That’s what happens
with the big ones. And as a consequence
of this, plasmids are what we prefer to work
with when we want to introduce a new DNA into a bacterium. And I’ll talk about that next
time, but what I want to show you here is how we make that
DNA to put into a bacteria. Here’s a plasmid. It’s a circle. If I want to put
a DNA into there, I’ve got to put a
break in that plasmid to put in the foreign DNA. If the plasmid that
I’m working with has a single site for
EcoRI, for example, I can cut that plasmid
with the EcoRI, and I’ll generate a structure
just like you see right here. It’s got a gap. Well, it doesn’t stay like this. The ends flop around
and everything. We’re showing in a
circular form to give you an idea about what
it looks like, but in fact, the ends
are anywhere there. If we take that circle and
we combine it or mix it with a foreign DNA that’s been
cut with the same enzyme– so let’s say I took human
DNA and I cut it with EcoRI. So now I’ve got
EcoRI-cut human DNA, and I’ve got
EcoRI-cut plasmid DNA. If I mix them together, and I’m
very careful about how I do it, and I put some DNA
ligase in there, I will have made
a recombinant DNA. That recombinant DNA, I
could put into a bacterium. And that bacterium
will replicate that and make useful
things for me, if I’m clever about what I’m doing
with it or how I designed it. So I’ve just made
a recombinant DNA. Now, I’m going to jump
ahead and show you something that’s a modified plasmid. Plasmids occur in nature. They’re not man’s inventions. But we have manipulated
some of those plasmids to be useful for us. So the last thing I’m going
to tell you about today is how we’ve manipulated
those to use them. What does it take to make RNA? Do cells just
automatically make RNA? What do they need? They need an RNA
polymerase, but what does the RNA polymerase need?>>[INAUDIBLE]>>What?>>[INAUDIBLE]>>Sigma factor for what purpose?>>[INAUDIBLE]>>Needs a template, but need
to have a promoter, right? So if I put a DNA
into a plasmid, and that plasmid
doesn’t have a promoter that RNA polymerase
can recognize, I’m not going to be
able to make any protein corresponding to that gene
I’ve just put in there. So one of the things
I need if I want to make proteins
in a bacterial cell is I need to have a promoter. So an ideal plasmid is
called an expression vector. What you see on the screen
is an expression vector. The first of those factors
that’s needed is a promoter. Second of the
factors that’s needed is we have to have a
replication origin. Well, since it replicates,
we know it’s there, but of course, we have to
have a replication origin. Those two things are critical. Now, I’m not going to talk
about the last critical thing until tomorrow. I think we’ve gone
through enough already. But I do have a relevant
song for you to sing, and it’s about translation. It’s fairly easy to sing. It’s called “Good
Protein Synthesis,” and it’s to the tune of
“Good King Wenceslas.” (SINGING) Amino acids cannot
join by themselves together. They require ribosomes
to create the tether. All the protein chains get
made according to instruction carried by m-R-N-A in
peptide bond construction. All subunit starts it
all with initiation, pairing up two RNAs at
the docking station. Shine-Dalgarno’s
complement in the 16 esses lines the A-U-G up
so synthesis commences. Elongation happens
in ribosomic insides where rRNA creates
bonds for polypeptides. These depart the
ribosome passing right straight through
it in the tiny channels there of the large subunit. Finally, when the sequence of
one of the stop codons parks itself in the A site
synthesis can’t go on. P-site RNA lets
go of what it was holding so the polypeptide
can get on with its folding. All right, guys see you Friday.