Introduction to Chemical Biology 128. Lecture 08. RNA.

>> The midterm one key has been
posted to the course website, and hopefully, you've had a
moment to take a look at that. Your scores have also
been posted and– >> We sent it [inaudible]. >> On Friday? >> Yeah. >> OK. And it was
sent off to the– Your exams are sent off to the
scanning service on Friday. So, I'm expecting
sometime today to be able to distribute them
back to you, OK? And as usual, they'll get
distributed back to you as PDFs that you'll download in
your MyEEE website, OK? So, everyone should get their
graded exam some time today.

OK. We'll talk more about
the exam in a moment. The journal article
report due February 14th, that's a week from Thursday. Double check these
instructions, OK? So, before you get
too involved in it, make sure that you've chosen
an article that conforms in the instructions because
again, if you haven't, I'll just give it back to you
and ask you to do it again. And that would really
be a bummer because you would have
wasted a lot of time, OK? So, double check the
instructions before you get too involved, before you go
too far down the road, make sure that your
article conforms. All right, let's get
back to the midterms. So, overall, I think this
was an acceptable job, it wasn't spectacular
but it wasn't the worst. Average of 56. Here, approximately is where
I put cutoffs for A minuses, for B minuses and C minuses. Note that I'm not looking to
give very many D's or F's, OK? So, at the very bottom,
if you kind of cling on and do an acceptable job on
your proposal and your report, it's unlikely that I'm
going to be failing you, OK? Now, I realize looking you
up today that the people who are probably at this end of
the bell curve might not even be in the classroom
today and that's fine.

But maybe they'll be
watching on YouTube. In which case, I have a
special message for you. You need to pick things
up a little bit, OK? I'm not looking to fail you
but I do have some standards and down here is below
what I'm expecting for an upper division
class here at UC Irvine. Now, I realize the
exam was challenging and my exams tend to be. So, roughly if I have to assign
grades tomorrow, about 29 people or so would get an
A minus or higher. And then, somewhere a
little bit below the– significantly below the average. The average is around
56, so that's over here. So, somewhere in here, I would
assign the B minus cutoff and then a whole bunch of people
will get C minuses or above, OK? So, something like that is
how I assign grades if I have to do it tomorrow, OK? All this is subject
to change though, OK? I just know that from
experience teaching this class that everyone wants to
know whether they got an A or B or whatever, OK? So, hopefully, you've already
gotten your scores back.

They've already been posted to
EEE, so you can look at this and get an idea of how
you're doing in the class, overall in terms of
a letter grade, OK? Now, if you're not happy with
how you did on this exam, come see me in my
office hours, OK? Stop by my office hours. We'll talk about your study
strategies and we'll try to come up with something that works
for you that hopefully takes you out of this, you know,
problem area that you're in, doesn't make you happy. OK. Speaking of which,
office hours, I'm going to have my floating
office hour for this week today, noon to one in the usual spot. My regular office
hour will be Thursday, 11 to 1 in the usual
spot as well, OK? So, I have two hours
office hours, the TAs have two
hours office hours, usual times, usual places. So, no changes there. Come by and say hello.

Talk to us about your exam. Tell us what went well,
what didn't go so well. I'd be interested to hear that. I'll also give you
an opportunity to provide some feedback. I haven't worked out the
details, but I'll probably set up some sort of survey of some
sort to hear back from you about how the class is going. It's week 5 and I'm
always curious to know how the students
feel about the class. So, anyway, there'll be a chance
for you to give me some feedback and we can do some midcourse
corrections if necessary.

I'll probably set that
up sometime this week. And if you do it, you'll
get some very minimal amount of extra credit. We're talking like
one or two points, OK? But one or two points,
better nothing. OK. Now, sometime else that
comes up around this time of year is several
of you will ask me to write a letter
of recommendation. I write about 100 to
120 letters a year of recommendation
for my students. It's something that
I take very seriously and here's what I've learned after many years
of doing this, OK? I've learned that you can
actually control the quality of your letters of
recommendation, that you can actually almost
orchestrate a good letter, OK? Now, the first thing
you need to is get to know all of your faculty.

You just never know who it
is who's going to be writing that crucial letter for you. It's just impossible to predict. So, good rule of thumb is
simply get to know everyone, OK? And the reason is many of
the programs and fellowships and other prestigious things
that you're going to be applying for are going to require
more than three letters, OK? So, you're thinking I have my
three letters all picked out. But no, suddenly you
need another letter. And so, you know, at the last
moment, it's really too late to find someone to write that. So, let's just assume
in advance that everyone of the faculty here at UC
Irvine would potentially be so and that you're going to
turn to for a letter, OK? So, simply get to
know all of them, OK? And if you're not coming out to my office hours
already, start now.

The other thing is once
you get to know everyone, choose your letter
writers carefully. You want to choose people who
are going to do a good job, OK? Now, all faculty are
totally vested in this. And you especially don't
want letters from, you know, your dentist site, OK? And no joke, I chair
the Admissions Committee and Department of Chemistry. In fact, I've been on this
Admissions Committee for going on 13 years, and for a while, I was also an undergraduate
Admissions Committee which gets 60,000
applications a year. So, I know a little
bit something about reading letters
of recommendation. And no joke, I saw one
from someone's dentist. And at the end, it
closed by saying, "This person has a
wonderful oral hygiene, and I really encourage you to
accept them into your program." But I can tell you as,
you know, as a lot of– as their oral hygiene
was it wasn't persuasive, to me that this person deserves
a spot in a PhD program, OK? So, you need to choose
people really carefully.

And I'm also telling you
this because when I look back when I was an undergraduate, I want to kick myself
and my own naivete. I was having, you know,
professors of English send in letters for, you know,
PhD graduate school. It's a miracle I eve got in. That's not the way
it works, right? It's better if I had gone
in my science professors to send letters for
science graduate school. Unfortunately, at least I got
someone in sciences to do that. But the point is that you have to choose these letters
appropriately. You want to choose people who
are going to take the time to care about you and invest
some of their own time.

It takes me about half an hour
or so per letter that I write. And so, it's a significant
investment of my time, but I'm willing to do it. You need to find other faculty
that are along those lines, OK? That are willing to invest
that amount of time in you. OK. So, choose the
letter writers careful. The next thing, get
involved at UCI.

I need your help, OK? I cannot write a letter for
you that's totally glowing and it just says, you know,
this person is a genius in chemical biology and
likes to play basketball with their friends
on the weekends, OK? And no joke, I've
written letters like that. But that letter didn't get the
person into medical school, and that's not what I want. I want to help everyone of
my students get, you know, hit their career goals.

And the only way I know to do
this is to get involved at UCI. You need to be doing something
well beyond the ordinary. You need some way of
distinguishing yourself, OK? If you have a four-O GPA,
you also need to do this, OK? Four-O GPAs are I
hate to tell you this. They are really common
at the level– people applying to
MD-PhD programs. There's a lot of
people who apply to PhD programs before
MGB [phonetic].

So, you need some way of
differentiating yourself. And what I'm talking about
is to differentiate yourself in some way that's
extraordinary, OK? And what I mean– examples
would be, you know, flying to, you know, the border with
Myanmar during a refugee crisis. And, you know, with a bunch
of people setting up a tent and starting to, you
know, do something, OK? That's dramatic. That gets people's
attention, OK? That's what gets you into say
a public health program, OK? So, that kind of thing is
what elevates your application overall the other
applications of people who have very good GPAs coming
from very good universities.

And so, now is the
time for you to start. And I look at it and I see some
of you just holding your heads, what's going on, no, relax, OK? It doesn't have to be, you know, something worthy
of a Nobel Prize. It doesn't have to be, you know,
something that's just, you know, totally ear fluttering but it
has to be something that come from within you, something
you're passionate about, something that shows
evidence of altruism, something that shows an ability
to go well beyond the ordinary.

That's what gets you into
really top programs, OK? And that's a sort of ammunition
I need to help you get into those program, OK? So, if you're not doing
this, start doing it now, OK? Along those lines, I'll help
you by pointing out some things, but come to my office hours
and tell me what it is that you're interested in and I'll see whether I can
create those opportunities for you, OK? Whatever it is, I'll try
to hook you up with someone who will help you
get that opportunity, but you need to start now. OK. Now, the next part
is you really need to give a long advanced notice. There's just nothing
that works– I can't do stuff last
minute and I don't think any of the faculty here can. We're all totally
oversubscribed. It's like a 100-hour week job to be a faculty member
here at UC Irvine. And so, if you come to me at
the last minute, you know, as, you know, as much as I might
like you and as much as I want to help you, it might
be totally impossible, because I might have five
other demands on my time that also have to
have deadlines.

And so, I just can not
do anything about it in circumstances like that. So, 68 weeks is kind
of a rule of thumb. If it's four weeks, you're kind
of pushing it but that's OK. But, you know, less than a week,
I'll just have to, you know, shrug and say I'm really sorry. OK. Now, I'll have some
paperwork that you need to bring to the meeting. It's really essential that you
get everything put together in a folder and I'll
tell you where to find that information about that. You can go to the website and then here's a
PDF that you can use. Use this to guide the
paperwork that you put together.

All that stuff needs to be put
together before your meeting. Send a gentle follow-up if the letter writer is
not heading your deadline. Sometimes, they just forget, OK? So, I tend to write all
my letters immediately. We meet and then we– I write
the letter within a day or so. But other people kind of
put them off on their desk and it gets buried
onto other stuff. So, feel free to send
a gentle follow-up. You know, it's carefully
oriented. This is really essential. It's very important that
you write a thank you note for every letter writer
that to every letter writer who supports you. Doing this provides evidence
that you're that classy person that they went to bet for
that they put their goodwill on the line for, OK? They were willing to tell their
colleagues, this is someone that I really like and they
want some affirmation of that, immediately after the letter.

The other thing is
it's very likely that this will be the last time
that you go back to the well and go back to that same letter
writer and ask for a letter, another letter, right? Who knows? Maybe you get into your dream
pharmacology program and then, you need– you want to apply for
a fellowship two years from now. And suddenly, now, you need to
go back to that letter writer, it would be helpful if you
had already sent them a thank you note. Now, this thank you note
should be handwritten. Don't use e-mail for
thank you notes, OK? E-mail is fine but in the bigger
world, e-mail doesn't, you know, or text messages is not
appropriate for thank you notes.

Thank you notes are
supposed to be personal. They're supposed to
tell something about you and one way you do this is
by showing your handwriting and showing that you're
taking a little bit of extra time and care. Now, the note itself
doesn't have to be on super fancy stationary
with your initials, you know, custom embossed on it, OK? So, it doesn't have
to be some engraved, you know, thing of beauty. All you have to do is take
a nice piece of paper, fold it in quarters and
then write something on it. That's it, OK? But that mere act of doing
that shows a lot of class and it again provides evidence that the letter writer was
right to take a bet on you.

OK. The last thing is along
those same lines of trying to make sure that the letter
writers are still in your camp when you need them years from now is keep the
letter writer informed. I will tell you that this is one of the great perks
of my job, OK? There's a lot of things I have
to do that I really hate to do, like fail students
and stuff like that. I hate that aspect of the
job, but what I love more than anything is hearing about
the successes of my students. It might be the only
perk of my job, in fact, now that I think about it. But it's an amazing
thing for me to find out that my students have gone
on to do something really cool and are rolling back to
frontiers in their own fields. I love hearing that. I love sharing in their joy and offering them
my congratulations. This is really essential, OK? This kind of closes the loop
and it's the payback to ensure that other people will
also be able to get– depend on letters
from that person, OK? That's the kind of– That's
the payment for doing this, OK? And along those lines, speaking
of payment, you don't have to provide gifts for
your letter writers.

In fact, it might be
better if you didn't, OK? And I think I have a rule on my
website, nothing over 25 dollars because I know I have a lot
of students whose cultures, tell them that it's
really essential that they provide a gift
for a letter writer. That's fine. I can respect that
culturally but nothing greater than 25 dollars and
I like chocolate, OK? So, it's really– So, OK? So, anyway, you don't have to
provide a gift but you do have to provide an update,
showing when you're successful because that's a
form of payment.

That really is why we do this. That's why I write letters
for 100 people a year. I like hearing about
their successes, OK? And I love knowing that
my students have gone out to do something
really cool, OK? So, I want to hear that. I want to hear that feedback. That really matters a lot to me. OK. Now, speaking
of letters from me, here is specifically
what I'd like you to do. Take a look at the guidelines
that I posted to the link on the course website.

That's over here. This gets updated
periodically but this is more or less the link that you need. Read this carefully
before you contact me. Again, request these
letters sometime in advance. And again, do anything
notable at UCI or beyond and then introduce
yourself to me, OK? You don't have to get to
know me in some deep way. We don't need to know each
other beyond this class. That's perfectly acceptable. In fact, that's the
way it usually is. But I just need to know
who it is that I'm going to be writing a letter for. I want to know that person. I want to know–
I want to be able to recognize their
name at some level, OK? Now, I will tell you that I
also write a lot of letters for people whose names– I
have no idea who they are, but I look at the course roster,
the grade roster and I noticed that they got an A minus
or higher in my class and I'm therefore able
to support them, OK? Now, in order to do that, I
will interview that person for like 15 to 30 minutes
before I write the letter, OK? And then during that time,
I'm going to decide whether or not I really want to
write the letter, OK? So, at that point, you
know, I'll give you a shot, even if I don't know you,
even if I don't know whether or not I should support you.

I'll do it just because
you did well in my class. And then, it's up to you in the
interview to kind of, you know, tell me why it is that
you really are passionate about podiatry or whatever it
is that you're applying for, OK? Whatever is that you're applying
form, I'm willing to get behind and get on the wagon and
support it, but you have to give me some reason
for that, OK? And it's likely that you
wouldn't have that reason today because you don't know what
you're going to be applying for, you know, a year and
a half or two years or three years from now, OK? So, that's fine.

I understand that. Don't panic. I'll be there when
you need it, OK? But again, check and look at
the guidelines ahead of time. OK. Any questions in
advance about that? Hopefully, this was helpful. I realized we took a lot
of time out of lecture but the truth is these types of
things actually are as important as chemical biology to ensure
your success beyond the classroom at UC Irvine. Questions? Questions? All right, good luck
on your letters. I will tell you that when
I review applications for graduate school, the
very first thing I do is flip up into the letters. I decide whether or not
I should read the rest of the application
based on whether or not the letters
are glowing, OK? To get into UC Irvine for
graduate school, we're top 10 in our program, letters
got to be really glowing.

And so, I'm looking for those
glowing letters even before I start looking at
the grades and GRE and all that other stuff, OK? All right, enough about that. Let's talk about what
we saw last week. So, last week, we wrapped
up the topic of DNA. And the most important
thing I can tell you about DNA is it's really
a superb nucleophile. This is as its essence
why DNA is so reactive and this is why DNA
is so susceptible to environmental affronts, and
we learned a lot about these. These are different
electrophiles that can then damage DNA, OK? And these electrophiles are
ubiquitous in our lives.

These are things like pesticides but they're also
innocuous things. Water, not an– is
not an electrophile but it can also damage DNA and we talked a little
bit about that. X-rays could damage DNA. Radiation from UV light, from
the sun, from cosmic rays, radiation cosmic rays, all
those things damage DNA. And so, we also talked about
how DNA can be repaired by excision repair.

OK. So, this damage the
DNA can corrupt its message and then interfere with normal
replication and transcription. We also talked about how
mutations to genes associated with tumor suppression like
p53 were especially dangerous for the cell because these
mutations to these genes that result at cancer oftentimes
termed oncogenes is required for tumor formation. And that's why they're seen,
mutations to these genes that then encode these
proteins are associated with such high percentages
of cancer or cancer has high percentages
of mutations to these oncogenes, hence the name oncogene
where onco means cancer. OK. So, we also talked about how
cancer results from alterations to DNA of the growth
factors, the tumor suppressors and cell division
checkpoint pathways. And we had to have mutations
to all three of those pathways. In fact, we found that
there are mutations to literally thousands, you
know, hundreds to thousands of genes before tumors really
started to take off, OK? And we characterize these
three pathways in the cell as being the accelerator, that's
the growth factor, the break, that's the tumor
suppressor and the clutch which is a cell division
checkpoint pathways.

And so, really cancer is a
really heterogeneous disease. It involves lots and
lots of mutations. It involves mutations to
all three of these pathways, which is mandatory for
the cancer to progress. And hopefully, you have a better
understanding of why it is that cancer rises and even
some strategies for helping to avoid damage to
your DNA and hopefully, hopefully avoiding
cancer yourselves.

OK. So, any questions about
any aspect of DNA, cancer, anything like that,
anything we saw last week? OK. Great. Let's talk next about RNA. So, last time, I introduced you to the multifarious
roles of RNA. RNA is really [inaudible]
as a biopolymer and that it really plays
a very wide range of– provides a wide range
of skills for the cell. We talked for example about
RNA acting as a catalyst to cleave other RNA sequences. We'll see some examples
of those today. This is a property that we're
going to call that we'll refer to as coming from a class
of RNAs called ribozymes.

We've already seen the idea
that RNA can deliver messages in the sense that
mRNA encodes proteins. It is translated. In addition, we're going
to see ribosomal RNA, this rRNA which is not monster
catalytic machine shown here in blue, where the RNA is
providing the essential catalytic function for
translating RNAs into proteins, for synthesizing proteins. Finally, we're also going
to talk a little bit about transfer RNA or tRNAs which deliver amino
acids to the ribosome. These are shown here. They're going to
deliver aminoacyl– Aminoacyl tRNAs are going
to deliver the amino acids to the ribosome in an
activated state appropriate for forming an amide bond during
peptide and protein synthesis. OK. So, that's kind
of the big picture. Let's dive in and
then start looking at the structure of RNA first. First, there are some important
differences between RNA and DNA. Most important is the presence of this 2 prime hydroxyl group
highlighted in green over here.

The second one is that in place
of thymine, RNA has a uracil. The only difference here is at
the carbon that's highlighted in green, thymine
has a methyl group. And I hope by the end
of today's lecture, it will be apparent chemically
why it is that DNA evolved to have a methyl group here in
place of just no methyl group, in place of a hydrogen, OK? OK. So, consequences of this
are pretty profound, right? The first consequence is that this 2 prime hydroxyl
provides an intermolecular nucleophile to attack the
phosphodiester backbone of the RNA. We've already discussed
this mechanism. I'm not going to belabor it
today, but this is, you know, this is a mechanism
that takes place at a fairly low rate
at pH 7, OK? So, a pH 7, this isn't
much to worry about, OK? When the pH comes off of 7 and
we already talked a little bit about the half life of RNA.

It's surprisingly long, OK? The real problem is when the
pH comes off of 7, you know, pH 9, let's just say pH 9. PH 9 will deprotonate
this 2 prime hydroxyl, making this an even better
nucleophile as shown here, OK? And that in turn leads to
much faster degradation of the RNA, right? Similarly, low pH can start to protonate the phosphodiester
backbone and start to encourage this nucleophilic
attack by the 2 prime hydroxyl. The other thing,
the real reason– Well, the other thing is that although RNA has very
long half lives, when we work within in the laboratory,
we really don't think of it as having a very long half life. In practice, it's
really kind of a pain in the neck to work with RNA.

And the reason doesn't have to
do with the chemical stability of the RNA backbone
rather it has to do with the ubiquitous nature of a
class of enzymes called RNAses. So, RNases are charged
with hydrolyzing RNA, OK? And they look something
like this. This is in black is the RNA
that it's going to digest. These are really
literally everywhere, OK? They're on your skin. They're falling off your skin. They're in your saliva. They're coming off of
your, you know, hair. They are really all
over the place and so, you're bathing the sea of RNases that your body naturally
secretes as a form of antiviral defense. It's a defense against
viruses because as you know, there are many viruses
that are RNA based. And so, if any of those
viruses happen to find their way through your skin,
hopefully, they're going to get digested very quickly
by any RNases that are present. And so, in practice, this
really makes it tough for us to work with RNA. And I'll talk to you a little
bit more about some strategies that are used in a moment.

OK. Before I do, let's
look a little closer at the mechanism of RNAses, OK? So, to understand
this mechanism, I need to tell you a little bit about the properties
of imidazole, OK? So, this structure here
is called imidazole. This is found on
histidine side chains and proteins like this one, OK? Now, here's what I want
you to know about this. The PKA of this nitrogen
over here of this protonated
imidazole is about 7. So, that means at pH 7, there is
a one to one ratio of protonated to unprotonated imidazole
present, OK? This really gives this
particular side chain some unique properties, right? PH 7 which is the pH that life
is taking place at or life in our cells, more or less life
in our cells is taking place at means that your imidazole
side chains will be 50 percent protonated and 50
percent unprotonated, OK? And so, this is really useful.

This means that this particular
functionality can either act as a base. In this case over here or
an acid over here depending on whether it has a
proton present, OK? So, it's very easy for
the protein to change around the charge state of
this particular functionality and it turns out that's crucial
to almost all of its functions in enzyme– as a catalyst. OK. So, let's look
in greater detail. This is now zooming in. Here is the mRNA or let's just
say the RNA to be digested. Again, here's the
phosphodiester backbone and there are two imidazole
functionalities found in this active site. One of these is deprotonated
to act as a base to deprotonate the
2 prime hydroxyl, making it a better nucleophile. And then, one of
these is protonated to protonate the
phosphodiester bond and help make it a
better leaving group, OK? So, the two of these
can work in concert. Meaning that there is a
single step to this reaction where on one side, a
proton is extracted. On the other side, a
proton is delivered.

And this all happens
in a single step, OK? And it looks, so the transition
state is going to look like this where the dash lines are
bonds that are either forming. And in this case or
breaking in this case, OK? So, this dash lines are all
the bonds that are forming and breaking in that
single step. OK. Now, I know what
you're thinking. You're wondering how it is that we know this is
just one step, OK? And actually to be up
until a couple years ago, I didn't have a good answer
to that, but I do now, OK? So, we can actually
now look at molecules at a single molecule level and then watch how many
intermediate steps there are by looking at distributions
of different rates. So, there's actually a
technical way of looking at whether something has
one step or multiple steps. In this case, we find that this
thing has just one step, OK? Which means that all of
the deprotonation and all of the protonation
is taking place in one smooth single
step motion. OK. And that's kind
of amazing, OK? So, we call this a
concerted reaction or concerted transition state
that allows this reaction to precede smoothly, OK? And we've already talked
about the strange intermediate which then very quickly
gets hydrolyzed to yield the broken apart RNA.

OK. Any questions about this? I have a question. What's up with this ammonium ion in the active site that's
donated by a lysine side chain? So, it's a primary amine
that's been protonated. Anyone want to pause it– propose a possible role
for this [inaudible]? Yeah, Chelsea [assumed
spelling]? [ Inaudible Remark ] Good. Yeah, so Chelsea
is reasoning that this phosphate group
is negatively charged. It's going to be
a leaving group. It would be such a
better leaving group if it was stabilized
by a nearby lysine, this nearby positive charge. And that's in fact
exactly what it does. Here's the buildup of negative
charge around the phosphate.

That's a delta minus
here delta minus here. So, having the nearby positive
charge to suck away some of the negative charge off
the phosphate helps stabilize that intermediate and lowers its
energy and then doing so helps to catalyze this reaction. So, really, in this active site, there's three functionalities
that are crucial. There are three functionalities
that are doing something to make this reaction possible and accelerate it to
an amazing degree. OK. Now, I told you earlier
that this is a serious pain in the neck for those of us who
work with RNA in our laboratory because the RNAses are
totally ubiquitous. They are constantly
attacking the very biopolymers that we're trying study. So– Oh, and I could tell you that we can find this RNA
stuff in the water, OK? So, you open the tops on your
shower, something like that.

The water that comes out will
have plenty of RNAses available to start chewing apart
any RNA on your skin, start chewing apart any
RNA that you happen to have in your reaction test tube. So, what we do is we
treat all of our water with a reagent called
depside, OK? Which looks like this. This is DiEthylPyroCarbonate,
OK? So, here is the RNase. And when you treat this depside, one of those histidines
are carbamylated, meaning that you switch them
from having a free nitrogen with a lone pair on it to now having a carbamate
functionality, OK? And notice that this reaction
is driven by the release of two carbon dioxide molecules. And I think this is actually
a pretty good reaction. I don't think the mechanism
is in the book, right? OK.

So, maybe I can ask you guys
Kritika and Mariam to go over it in discussion section
this week, OK? Thanks. OK. This is a good reaction. But again, this business about
driving the reaction forward by release of carbon
dioxide is a time tested and a very viable mechanism
for a very viable strategy for accelerating reactions. Because remember, this carbon
dioxide is going to go bubbling out of the reaction flask, in
this case, a big bag of water. And the result will
be a reaction that gets driven forward by
Le Chatelier's principle, OK? By the fact that this
carbon dioxide bubbles away, preventing the reaction
from going in reverse, OK? So, this is a very
effective reagent.

The other thing we do is
we then take this reagent and make a solution of it
and start wiping down all of the surfaces in
the laboratory. And then, we're really
rigorous about wearing lots of gloves and, you know, very
carefully putting on, you know, quotes that have a big
contaminated RNAses. And in fact, if in my lab,
if we end up doing this, we'll actually designate
one bench of the lab that no one is allowed
to even step near, OK? Because little tiny, you
know, spit blobs and stuff like that would be enough to
ruin the whole experiment. So, no one is allowed to
even look at the bench, OK? It's set off in a corner
and I ask everyone, not even to walk
near the bench, OK? But that's the kind of
paranoia that ensures that your experiments with
RNA go well, 'cause otherwise, this starts to degrade or that
drives you absolutely baddy, OK? All right, let's get
back to structure of RNA. RNA as we discussed
earlier has a 5– lacks to 5-tri-methyl of thymine so it has a uracil
in place of thymine.

So, it has a uracil
in place of thymine. Now, let's talk a little
bit at why DNA evolved to have this extra
methyl group, OK? So, all DNA, here is a
structure of DNA is subject to hydrolysis by water. And I don't remember
if we mentioned this when we talked about DNA. Does anyone recall if
we'd mentioned this? Yeah, I don't think I did. I actually think I took
it out of the slideshow. In any case, water can hydrolyze
this cytidine base over here, removing, replacing
the amine functionality with now a carbonyl
functionality. And this should not
be a huge problem, OK? So, in DNA– Oh, by the way,
the result here is a uridine.

Notice that this– the
result switches the pattern of hydrogen bonding. Over here, it goes
acceptor acceptor donor. And now, it goes
acceptor donor acceptor. Big difference, right? Again, it's AAD and
over here, it's ADA. OK. And that's a big difference. That means that you're going to
get inappropriate base paring. That means that replication
of DNA is going to give you the wrong sequence. Transcription will give
you the wrong sequence, OK? So, in DNA, there are enzyme– There are repair enzymes that
are constantly circulating and looking for examples
of uridine. This is uridine in the DNA. And when they encounter
them, they will replace that uridine back with the
C, back to the correct base, because they know that that's
the one base that's susceptible to this hydrolysis, OK? And this happens at
a fairly fast rate. I think the half life is
somewhere on order of a year or something like that. So, one year– This just means that pretty much all
the C's [phonetic] in your body had been
replaced a couple of times at this point in your life.

And so, without this mechanism, the code would have
been scrambled around a long time ago, OK? So, this is essential really
for life that's based on DNA which is all the life
on this planet, OK? So, the shorter lived RNA
however does not need this code. RNA doesn't have to hang around
for years upon years, right? And so, for this reason, RNA has
evolved with just using uridine and place of thymine but DNA which has a much longer half
life really does need this mechanism for repair. Does that make sense? Questions about this concept? OK. I always get nervous when
I ask if there's questions and no one has any questions.

You know, you guys–
Feel free to stop me if anything comes up, OK? All right, the other thing
in addition to the bases that I showed you, in
addition to this business about replacing thymidine with
uridine, RNA bases are subject to heavy, heavy modification. And I'm showing you just a
few examples on the slide. In blue, these highlight
the changes. And some of these changes
are relatively minor like this one used to have
a carbon-carbon double bond and now, it's a carbon-carbon
single bond. But some of these modifications
are, you know, there I say kind of radical, like
this one over here which has this oxyacetic
acid functionality appended to uridine. And these alterations to the RNA after it's been synthesized
drastically effect its function, OK? So, they control things like altering the
structure of the RNA.

They can also affect
the recognition. This becomes especially
important when looking at transfer RNAs, where
transfer RNAs are going to be delivering
these amino acids to the ribosome during protein
synthesis, during translation. And so, it's not easy to get
all of the encoding identity from this tiny little
amino acid. So, instead, the tRNAs
include different modifications that make these changes to the amino acid a
little bit more apparent to the ribosome during
translation, you know, just allows a little
bit extra testing or even a little bit extra
testing during the amino acid loading step, where the amino
acid is loaded on to the tRNA. OK. So, let's talk a
little bit about structure. First, here's the structure
of the tRNA that I alluded to on the previous slide, OK? And compare that against
the structure of DNA. So, the big difference here is that DNA is always double
stranded, at least, you know, 99.9 percent of the
time in the cell.

So, it doesn't have this
opportunity to fold in on itself and form Watson-Crick
base pairs on itself, rather it has a second
complementary strand that can form this
nice B-DNA structure that we've come to
know and love. On the other hand, RNAs
are often single stranded or typically single stranded. And so, for this reason, they can readily form
Watson-Crick base pairing as exemplified by
this region over here or this region over here. But in addition, they can also
form Hoogsteen base pairs. Little hard to see here but there's some non-canonical
base pairs taking place in the center of the structure. So, in addition to the
regular Watson-Crick base pairs that we've discussed
in the context of DNA, we're going to start seeing some
other non-canonical Hoogsteen base pairs, which I
believe we mentioned earlier when we talked about DNA.

So, we've already seen
these Hoogsteen base pairs and they come up more often. OK. And here's another
example of RNA. In this case, this
is two strands of RNA, forming a ribosome. And we'll talk– or
sorry, forming a ribozyme. This is an enzyme, a
catalyst that's based on RNA that has RNA as its
backbone, OK? So, wide variety of different
structures, you know, no holds barred, all kinds
of things are possible. In the structure of RNA, they
form all these twisty structures that are continually surprising. And that's one of the great
joys of working with RNA is that there's a large
capacity for seeing stuff that hasn't been
observed previously. OK. So, let's talk– Let's try
to systematize the structure and this is the same
nomenclature that we're going to use in the context of talking
about protein structure as well. So, I want you to get
comfortable of this idea of primary, secondary
and tertiary structure.

So, starting at the top,
the primary sequence or primary structure
of either RNA or proteins is simply
its sequence, OK? The listing of CGAT, et cetera. Oh, and if you look
carefully here, you'll see some other
weird stuff, right? Like this weird, you
know, Greek symbol psi in the middle of this sequence. That's for one of those
bases that I showed earlier where the base had
been modified, OK? And that base happens to be
essential for the structure of the resultant,
tRNA in this case, OK? So, there's a couple
of weird ones in here.

And again, there's a Y.
There's the psi thing. Those again are modified bases. Those are special cases
that had been modified, OK? So, that's the primary
structure, simply the listing
of its sequence. And again, we're always
going to try to list those in the 5 prime to
3 prime direction. So, if the 5 prime and 3 prime
are not depicted, you can assume that the 5 prime is on the left and the 3 prime is
on the right, OK? That's the convention that
will always be used for RNA. The secondary structure is a
listing of the RNA, how it folds up to form Watson-Crick base
pairs and Hoogsteen base pairs.

And when we take this primary
structure, we find that these– the yellow region
can form a loop and the regions nearby
can actually form perfect Watson-Crick base pairing
complementarity to each other, where if you look
closely at this, there's actually three hydrogen
bonds of the GC, two for the UA, three for the GC, et
cetera, going across here. And then, there's regions
that are called loops. There's these loops at the ends.

And then, there's
one loop in blue which is termed the
anticodon loop. That's the loop that's going to then hybridize
Watson-Crick hybridize to the mRNA during
protein translation, OK? Recall again that
the tRNA is going to deliver the amino
acid during– to the ribosome during
translation. That amino acid is
attached as an ester to the 3 prime end of the tRNA. OK. Last thought about
secondary structure. In this case, the secondary
structure looks a little bit like a cloverleaf, OK? That's shown here. And so, for this reason,
this is often called a cloverleaf structure. The one problem is that when we
actually look exactly what the structure looks like as solved
by X-ray crystallography and shown in two
views over here.

It doesn't look anything
like a cloverleaf anymore because it gets so
wrapped up in itself. It gets so twisted around
that it no longer looks cloverleaf like. But the cloverleaf
nomenclature has stuck with us. And so, we're going to refer to
this as a cloverleaf structure. OK. Again, down here
in this blue, this is the anticodon
loop that's going to bind to the mRNA, try to form perfect
Watson-Crick base pairing complementarity and if that
happens, that tells the ribosome that this is the correct
amino acid, way off here at the 3 prime hydroxyl, OK? Way up at the 3 prime
end of the tRNA. OK. Let's take a closer look
at secondary structures. I've shown you some examples. Helices are secondary structures that are forming
Watson-Crick base pairing, G's and C's, A's and U's. Loops or hairpins are
regions that are unpaired and allow turns to take place.

Let's take a closer look. So, in blue, in red, in yellow,
these are examples of loops. There's no base pairing that's
taking place but this form, you know, 108– This form
turns in the secondary– in the structure of the protein. And you could also have internal
loops where there's sort of loops crossing each other and then junction loops
where two strands. That might actually be
connected, join each other and lead to sort of, you
know, three arms leading away. So, there's really a wide, wide
variety of secondary structure.

Good news in this DNA, it's just
actually totally predictable. There are programs called
Mfold and other programs that will actually predict the
secondary structure of RNA. And in doing that by
giving you some insight and to what the tertiary
structure might look like, so you can actually just
simply type a sequence in and then get a pretty
reasonable approximation of what the structure of the–
what the secondary structure of the RNA is going
to look like. And those actually
work pretty well. They work by trying to
maximize the number– the stability of
the result in mRNA by maximizing these
Watson-Crick base pairs. OK. Any questions
about RNA structure? OK. The tertiary structure
is very hard to predict. It's amazingly hard to get
the stuff to crystallize. This does not like
to crystallize. The process of crystallization
of course forces it to get closely packed up
with other RNA molecules, RNA as a phosphodiester
backbone. It's negatively charged. It doesn't like getting
all packed together with other RNA molecules. So, structures like this one
are pretty far and few between.

We don't have nearly as many RNA
structures as we do of proteins. And again, that's due to
the fact they just don't like to crystallize
with each other. OK. Switching gears, let's talk
about the synthesis of mRNA. A lot of what we know is do–
really to some phenomenal work by the Kornbergs,
father and son. Roger Kornberg in 2006
won the Nobel Prize for determining a
structure of RNA polymerase. His father in 1959, here's
his father won the Nobel Prize for studies of DNA polymerase. I've actually met
Arthur Kornberg, the father over here
when I was a postdoc. And it was a group of
postdocs that met with him. He actually carried this
book with him like one of those blank pages books and
asked everyone who met with him to sign the book because
he wanted to keeps track of everyone he met with
in his entire life, which is I thought was kind of
an eccentric but touching hobby.

So, I don't know exactly
what he's doing at, you know, and to a haul of signatures. I mean I can't imagine anyone
going back and looking at– Oh, yeah, I met him back
when he was a youngster. I mean it's really,
it's sort of– Anyway, he was also
a fascinating person to talk to, really,
really smart. Unfortunately, he's passed away,
but really phenomenal scientist. OK. So, let's talk a little
bit about RNA polymerase. We'll talk a little bit more
about the structure in a moment.

This is kind of a crude
overview of the structure. In short, this is the enzyme
responsible for transcription of converting DNA into RNA. And enzyme synthesizes
this new– this RNA in the 5 prime
to 3 prime direction. This is good news, right? That was the same directionality
that DNA polymerase used. And we talked about how all life on this planet synthesizes
in that direction. The other really curious
thing about this enzyme or the remarkable thing about this enzyme is it
doesn't rely on a primer. So, I talked about how DNA
polymerase always requires a primer and the importance
of that primer was to direct the DNA polymerase
to a specific spot in the DNA. RNA polymerase relies on
transcription factors, proteins that drag
the RNA polymerase into a particular region of
the DNA and get it started.

OK. So, let's zoom in
and take a closer look. So, this is a very
large enzyme, OK? And it looks a little bit like
a jaw, so the enzyme itself as it moves, as it makes it
to one of this bond has kind of a jaw-like structure, OK? So, it has a different mechanism
than the right hand structure that I told you about
of DNA polymerase. It looks more jaw-like. It makes a much more– a
much larger macro mechanism. Are you thinking
what I'm thinking? Yeah. OK. So, sorry, in joke. We were thinking that
this would be a good one to do some experiments later,
but we'll talk about it later. OK. So, back to the topic. In green, this is the RNA
that's the nascent RNA that's being synthesized. In red, this is the incoming DNA
that's providing the template for the DNA that's
going to be transcribed. And here's a zoom in view
showing how the RNA that's being synthesized that get in green
forms Watson-Crick base pairing with the DNA and then
it's kind of ejected out of the enzyme active site.

In addition, there's
a long tunnel through which the
nucleotide triphosphates which are the building
blocks of RNA travels to get to this active side up here. So, these guys are kind of being
sucked in through this funnel and they zoom in here and that's where they're actually
built, OK? More or less though,
the mechanism is more or less exactly what we
saw with DNA polymerase. It has some different kinetics. It has, you know, some
differences in terms of the shape of the enzyme. But it's more or less
the same mechanism, OK? So, everything I told you
about with the magnesiums, with the carboxylate, it's
chelating to those menus, everything we talked about last
time with DNA polymerase applies to RNA polymerase as well.

OK, except for the
major exceptions that I had been telling
you about today, OK? But the mechanism,
exactly the same. OK. Let's take a closer
look at transcription. So, during DNA replication,
their RNA is synthesized to act– Sorry, I'm not
talking about transcription. I'm actually talking
about replication of DNA. During DNA replication,
a little short stretches of RNA are synthesized at these
primers for DNA replication, OK? So, the enzyme DNA primase is
actually an RNA polymerase.

And again, the synthesis only
takes place in the 5 prime to 3 prime direction, OK? So, here's the parental
DNA in purple. That's going to be
replicated in red. This is the new strand
of DNA that's going to be synthesized, OK? And so, pulling thing– these two strands of DNA apart
are an enzyme called helicase and there's also some
single-stranded DNA binding proteins that get
in there and start to stabilize the
single-stranded DNA. DNA primase then comes along and synthesizes these green RNA
primers that then are extended by DNA polymerase during
polymerization, OK? So, questions? OK. Let's take a closer
look at DNA primase. In red, this is the
single-stranded template. This requires a short primer
to get polymerization started.

Oh, OK, so in this case, sorry. I'm losing track of things. So, transcription also depended
upon DNA primase as well to provide that primer. OK. Let's take a closer look
at transcription in action. So, transcription takes place
at multiple sites along the DNA. And I'd also– It evolves
not just, you know, one transcription of that
and then back to the start, rather it's sort of like
parallel growth of large numbers of mRNAs that are all being
synthesized and parallel and at the same time, OK? So, over here, this is
the one end of the DNA.

Here's the DNA. Here's one gene. Here's a second gene over here. And here are some small mRNAs that have already
been synthesized. So, what we're looking at is
we're looking at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, et cetera RNA
polymerase molecules zooming along here. And as they get further
and further along the gene, these RNAs that are being
synthesized get longer and longer, OK? So, this net result is
it gives you something that look a little bit
like a Christmas tree. So, this is an electron
micrograph that's an image that was taken using
an electron microscope. And to me, it's absolutely
spectacular, OK? So, I don't fault my
friends who uses image as– for their Christmas cards
because they really does look like a Christmas tree. And I think it's just absolute
spectacular image over here. OK. Let's zoom in a little bit. In order to get transcription
started, the DNA has to– the piece of DNA that's
going to be transcribed, the gene that's going
to be transcribed has to be identified using
transcription factors. So, there are two kinds of
transcription of factors.

There are– They all
involve proteins. So, some of them sit on a
promoter regions of the DNA. In other words, they
promote or accelerate or and some way encourage the
transcription of the DNA and then there's other sets– sit on regions called
terminators to shut down the transcription
of the DNA, OK? We're going to talk
next about the– how it is that these
proteins bended DNA and get RNA polymerase
to come along and start transcription, OK? So, many of these transcription
factors bend or some of them bend DNA into some
pretty dramatic shapes, OK? So, this involves, you know, bending the sequence
a 180 degrees around the transcription factor. The vast majority
or the majority of transcription factors
and have alpha helices that fit neatly into
the major group.

And we've talked
about this before. We've talked about how the
sizing is almost perfect for a alpha helix secondary
structure of a protein to fit into the major group. But that's not the only way that transcription factors
can find can bind to DNA. In this case for example, notice
that these are beta sheets, they're beta strands,
two beta strands that are fitting
into the major group. There's no alpha helix here. So, you can have
both beta strands and alpha helices binding
to the major group of DNA. Let's talk a little bit
about this bendiness. OK. The bendiness actually–
and here's an example, this is the TATA-binding
protein riding the double helix. It's described as
looking like a saddle. Here are the stirrups down
here, here's the saddle up here. The south of the
helix is the cowboy and it's actually grabbing
on to the DNA and forcing it into this bent configuration. This business about
forcing the DNA into particular structure
provides an additional level of sequence specificity, right? I guess recall earlier we talked about how DNA structure
was set by the pi stacking.

And the different bases, different base pairs had
different ability to pi stack in different stickiness
with each other. So now, we have this
transcription factor that's literally sitting a stride
the DNA and forcing it into a particular configuration,
thus testing the strength of its pi stacking
in its base pairs. So, in addition to the molecular
recognition of specific bases, which I'll describe in a
moment, this has another level of sequence specificity in terms
of just bendiness of the DNA, which again is a
sequence-dependent property. OK. So, not all sequences
will tolerate this affront.

And this is pretty
dramatic, right? This is a 100 degree
angle down here. OK. So, the transcription
factors decide whether or not they're going to bind to the DNA based upon
binding to other molecules. Transcription factors are
rarely operating by themselves. More commonly, they form these
large holo-complexes of lots and lots of proteins that
kind of pig pile on each other and then pig pile
on the DNA, OK? It's like a super
bowl Sunday except with two good teams
on the field. But here's some example
of what I mean by that. OK. So, in this case, this is a
transcription factor called the tryptophan repressor. And no surprise, the
name actually here is very descriptive. This is the– This is
the transcription factor that provides feedback for the
biosynthesis of tryptophan, OK? So, if the cell has too much
tryptophan being synthesized, the transcription factor
then jumps on to the gene and forces it to shut off. So, it shuts down the
synthesis of tryptophan. So, what happens is tryptophan which is an amino
acid can bind directly to this transcription factor.

When it does, these two alpha
helices here swivel, OK? So, they're going to
from this configuration to this configuration. And I know that seems very
small, we're just talking about a little change. But that change is enough to
allow out the alpha helices to now fit down into
the major group, where previously they
couldn't fit, OK? Previously they're like
this, the tryptophan binds, it pushes them into
other configuration and allows the binding
to take place.

OK. So, this stuff is
very tightly regulated by multiple binding events. When we take a closer
look at the interactions that are highlighted with this,
I don't know what you call them, echelons over here, OK? Let's zoom in. I want to talk to you about how
transcription factors recognize specific sequences of DNA. It would be bad news for example if the tryptophan
repressor starts grabbing on to random sections of DNA
and preventing the synthesis of a central housekeeping
genes for example. That would be a total
disaster for the cell. The cell can't have
transcription factors running around and shutting
off random things.

Rather, transcription factors
have amino acid side chains that recognize specifically
particularly DNA sequences. And here is the way this works. OK. So, in this case, I'm
showing you a DNA sequence and I've tried to simplify
it as much as possible, the ribose rings
are these pentagons, the circles are the
phosphodiesters of the DNA sequence. And the transcription
factor binds to the backbone of the DNA, OK? And that's shown by these
dash lines and arrows, OK? So, the arrows indicate hydrogen
bonding functionalities. And the dash lines
indicate other interactions. So, for the most part, the
transcription factor is grabbing on to the phosphodiester
backbone of the DNA and more or less riding along it, OK? So, I like to think of this
as kind of like a gymnast with parallel bars, OK? Or a train on railroad
trucks, where the trucks or the parallel bars are
the phosphodiester backbone of the DNA, OK? So, the transcription
factor and let's imagine that parallel bars,
it's easier to imagine.

OK. So, I'm up on
the parallel bars. OK. So, I'm the transcription
factor. I'm now going to be walking
along the backbone of the DNA. But I'm making the contacts drew
my hands on the parallel bars which are the transcription
factor interacting with the backbone of the DNA. Notice that there
are no interactions with the bases of the DNA, OK? And now, as the DNA
starts moving along this — Sorry, I said transcription
factor starts moving along this DNA sequence, it
looks for places where it side chains can form
interactions with the bases of the DNA, the base
pairs of the DNA, OK? Oh and by way, although
I'm using this analogy where it's parallel bars.

And I'm kind of studying apart. This thing is moving at the
speed of a freight train, OK? It's really conking. It's scanning through thousands
of bases per minute, OK? It's really, really moving
along at a fast, fast clip. And as it's moving along,
it dangles its side chains into these base pairs, OK? So, it's forming specific
contacts over here. But it's got, you
know, it has, you know, its feet running along
the bases over here. Kind of like a finger
moving along the piano, OK? So, it's moving along thousands
of base pairs a minute. Eventually, it finds bases
that have particular contacts that are complimentary to the
side chain functionalities of the transcription factor. And those are highlighted
by these arrows, OK? And notice now, we have lots
of arrows that are going to the bases in the middle
of the DNA where we switch from a nonspecific
complex, meaning, it's not recognizing a
particular DNA sequence to a specific complex.

There is a big difference in where these interactions
take place. Over here, they were
entirely on the rails, the phosphodiester bond rails
of the DNA, the backbone rails. And then over here now, it's found very specific
places to interact with. Furthermore, it turns
out that it doesn't make direct interactions. The transcription factor does
not make direct interactions with the DNA base pairs. Rather, it looks for an intermediate
water that's been set up in a particular
configuration, determined by the
sequence of the DNA. This is a very important
point, OK? And I want to repeat it
because it's so important. The DNA has ordered
water on its surface. There are water molecules
that are patterned by appropriate hydrogen bonding.

Donors, acceptors
and that water sets up a pattern on the surface. What these means then is that when the transcription
factor binds, it can actually read out
the sequence of the DNA by interacting with those
ordered water molecules. Meaning, that it doesn't have to push apart all
those water molecules that would be naturally found
on the surface of the DNA, OK? In other words, it
doesn't have to put in all of the energy associated
with that kind of effort.

This allows the transcription
factor to race along the DNA
sequence much more fast, much, much faster than with
otherwise be able to, OK? So, when it finally alights on
the correct sequence of the DNA, it can then form all of
these contacts highlighted by the arrows. And many of these contacts are
indirect, meaning that they're through waters that are bound
to the surface of the DNA. OK. Now, that's a little
bit of advance concept, any questions about that? Yeah? >> [Inaudible] kind of
explain, how does it move down– >> How does this
slip along there? >> Yeah, was it like
what drives– >> What's the driving force? OK, excellent question,
what's your name again? >> Shawn [assumed spelling].

>> Shawn. OK. So, Shawn's question is why
should I bother moving it along at all? Why doesn't it get stuck there? The transcription
factor is looking for a more stable situation, OK? So, when it's in this
configuration over here, you know, it's like me
on the parallel bars. It's not all that
happy up there, OK? You know, it's a
little uncomfortable.

And what it's looking for is
this looking for greater number of interactions that
stabilize it, OK? And so, it's really
thermodynamics that's driving it along, OK? So, what's it's looking for is
the net energy gain that it gets when it forms the perfect
hydrogen bond over here, when it forms lots
of hydrogen bonds, when it forms other contacts
with the DNA sequence and that's what's driving along. It's a question of equilibrium.

So, yeah, it can get stuck
over here but it's not all that happy being
stuck over there. And so, it's looking for
something more stable. So, great question, Shawn. Other questions? All right, let's take a
look at a few other examples of transcription factors. This is a class of transcription
factors called zinc fingers. They're named after the zinc
ion, zinc ion which is chelated between the two imidazoles,
check it out.

Here's the functionality that
we saw earlier in this lecture. In this case now, it's
forming the [inaudible] to a zinc 2 plus ion. In addition to two violates that
are also chelating to the zinc. Zinc in this case
is playing a role of structuring what would
otherwise be a very tough sequence to structure. This is a relatively
short protein. A relatively small
protein and so, it really relies upon
the zinc to help it, force it into this
particular confirmation. Otherwise, since it's very
small– small proteins, small– short sequences of protein
are often called peptides. Meaning that they're unfolded that they don't have
any inherent structure. In this case, the zinc
contributes inherent structure by forming covalent bonds
to specific side chains down in the protein, OK? Something that's notable
about this is that there's– these are actually two
transcription factors that are joined by a
linking sequence over here.

And so, the advantage
of the zinc fingers is that you can string them
together like beads on a string. And in doing so, you get
greater sequence specificity. Where this guy over here would
be reading out I don't know, say four or five base pairs. And then this guy up
here would be reading out an initial four or five. You start putting together a
bunch of those four or fives and pretty soon, you get
something at specific for the 18 base pairs
necessary to call out a particular zip
code of the genome, OK? And again, 18 is
the magic number where if you have something
that can specify 18 bases that can then be a unique
address within the human genome.

Zinc fingers are immerging
is a really exciting powerful technology for directly
manipulating DNA. And we're actually at that point
where we can sort of program in specific sequences
of zinc fingers that we know will then bind
to specific sequences of DNA. And in doing this, we can design
our own transcription factors and even do things like
bring along nucleus as to chop apart
specific sequences of DNA. And I've been waiting for this
my entire scientific career. But I don't think I'm going to
have to wait that much longer.

It looks likely– This
is a sort of technology that will make gene
therapy possible. Where we'll be able to go in
and fix our incorrect sequences of DNA using artificial
transcription factors like zinc– like designed
zinc fingers like this. OK. Here's another example
of a transcription factor. This is engrailed
[inaudible] domain. This is a transcription
factor from originally named for a particular phenotype and
fruit flies and Drosophila. Again, this has this
nice alpha helix getting into the major group. In this case, this
alpha helix access kind of like a barcode reader, OK? So, everything I
told you previously about riding the rails,
that's applicable here, OK? But the thing that's kind of tickling the piano
keys that's checking out the sequence is this major
alpha helix that's spinning neatly into the major group, OK? And like a barcode
reader, you know, if you're at Albertson's
over here.

And you decide to get that
option that doesn't have a line where you have to do
the scanning yourself. You know what pain in
the neck it is, right? You know how– You're
kind of there, you're kind of swiveling
the barcode back and forth. This relies upon a similar
swiveling kind of action, OK? Where the alpha helix is
kind of twisted backwards, forwards, twisted like that. And in doing that that then
checks the sequence of the DNA.

And we know that because if we
make changes to it over here, that affects how it swivels
and affects the specificity in the major group of the DNA. One last thought, this– you
see this little trailing part over here? That's the– one of the
termini of this protein and it's interacting just a
little with the minor group up here, and that makes it a
little bit unusual in terms of transcription factors. But we found that actually that little tailing piece is
not essential for the function of transcription factor. You can cut it away and the
transcription factor remains the same, OK? And by the way, those kinds of
experiments that I'm describing to you, you're chopping a
part transcription factors to check how they function. Those make for great
proposal topics. OK. This is one of my favorites. I'm a huge fan of just
collecting protein structures. I love this, these
reminds me of chopsticks. This is a leucine
zipper and it's named for the hydrophobic
functionalities found in the various center of
these two alpha helices that form a coil,
coil with each other. And they're held together by
losing these leucine side chains from a leucine functionality, simply a hydrophobic
functionality.

And these grips the
DNA like chopsticks. And with two alpha helices, one
interrogating this major group up here and the other one
interrogating this major group down here. And it is truly beautiful
structure. OK, let's put everything
together, OK? So, it turns out that
what I've been showing you with the isolated, you know,
chopsticks or this guy, that's not the real picture
of how transcription works. Transcription works again
by having large numbers of proteins pig piling on
top a specific DNA sequences. And that in turn can recruit
RNA polymerase and other factors that are necessary for RNA
polymerase to start cranking out RNA during transcription. And there's a lot
I can say here. I think I'm going to
keep it relatively short. Let me show you some
specific examples that are actually useful in the
laboratory of RNA transcription. OK. So, a classic and one that's
used very routinely applying small molecule control over genetic transcription
is the lac repressor.

So, the lac repressor binds the
DNA and then in the presence of lactose changes
its confirmation. And then is released
from the DNA and then that in turn turns
on expression, OK? So, that turns on transcription which in turn turns
on translation. And that combination of
transcription followed by translation we're
going to call expression because it means expression of
a protein from a DNA sequence. Now, here's the way
this works, OK? So, here's the structure
over here. And then in green, these
are the lactose molecules. So, here's what lactose
looks like, recall that lactose is a
disaccharide consisting of galactose and a
glucose held together by a beta glycoside
acidic bond, OK? It turns out that yes,
you can– you can– so this is used very
routinely in biochemistry and chemical biology
laboratories as a technology for turning on the expression
to specific genes, OK? So, for example, if
you want, you know, I'm just making this up, if you
want tomatoes that are more red or something like that.

You might want a gene
that can be turned on in the presence of lactose. OK. I don't exactly
why you want to do that but let's just say
that's your goal, OK? Or in my laboratory, maybe
you want to turn on expression of a particular protein that
you're then going to study in think greater detail, OK? That would actually
be a better example. So, in that case then you have
the DNA in black in the E. coli, you then add lactose, OK? The problem is, lactose has a
very short half life in cells.

It could be hydrolyzed for
example by beta galactosidase and to the two monosaccharides. And so, oftentimes
instead of adding lactose, we'll add a synthetic analog
to lactose called IPGG. And this has a bio
glycosidic bond in place of a regular glycosidic bond. So, it's no longer recognized
by beta galactosidase and has a much longer half life. Its half life is
still not great. It's hydrolyzed by
water very slowly over a course of a few hours. So, this, you know, it works but
it's not the world's greatest. So, the way we do this is
we grow the E. coli cells, we then dose in IPGG that in
turn freeze up the DNA sequence. And the DNA sequence gets
turned on and the protein of interest gets
synthesize for us, OK? We do this all the time, OK? And we're not alone in this.

Thousands if not hundreds
of thousands of laboratories across the country are
doing this as we speak, OK? So, this is a very
common technology. It's something everyone
needs to know about. A major goal of chemical
biology of course would be to exhibit this level of
control over specific genes. It would be a very cool if you
can add drugs, small molecules and give them to patients that
would turn on specific genes or shut off other genes. If we can do that, we
would basically cure many, many, many diseases. That would be very powerful. We're not there yet
but that's one of the– It's a major goal of a number
of chemical biology laboratories and also pharmaceutical
companies. OK. So, here's the other
thing that you can do with this particular
transcription factor. It turns out that
you can separate out the DNA binding domains
shown over here separate from the transcription
activation domain, OK? And you can then fuse
the DNA-binding domain to some sort of bait protein.

And fuse the transcriptional
activation domain to say a prey protein. And when these two interact with each other then you
get the functional complex of the transcription factor
allowing transcription down here, OK? So, these two, this interaction, this protein-protein
interaction is enough to bring together
the missing pieces and the restored
transcription factor can then initiate transcription. It turns out that this is a
very powerful test for whether or not proteins will interact
with each other whether bait and prey will interact
with each other. This is something as called
a yeast two hybrid system. So, it's a hybrid of these
two proteins over here. And it's used again
very routinely. Here's the way this works, OK? So, you do this experiment
where if the protein is– if the proteins can
interact with each other, it turns on transcription
of the gene that encourage
beta-galactosidase.

Beta-galactosidase in turn
can hydrolyze this indoxyl l. And indoxyl can then
spontaneously dimerize to form in deep indigo blue. And I see if you could go–
wearing their jeans today. Blue jeans are dyed using
this dye right here, OK? So, that stuff precipitates
out a solution and forms an intense,
intense blue. And you can actually
see that here, OK? So, clear colonies
like this, no binding. Blue colonies, that equals a
protein-protein interaction, OK? Because that means that bait and prey are interacting
with each other. And then in turn means that
we're getting transcription to result in over expression
of the beta galactosidase gene. OK. Now, so far, I've been
showing you some pretty simple examples. It turns out that things
get a lot more complex when we start looking at
eukaryotic mRNA processing. And I think what we'll
do is we'll stop here. When we come back next
time, we'll be looking at all the ways mRNAs are
modified during eukaryotic transcription and translation.
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