[ Silence ]
>> OK, welcome back.
We're back.
We're going to pick up
where we left off last time.
Last time we were talking about
RNA and all things RNA related.
In particular today, we're going
to be talking about translation
of messenger RNA
to make proteins.
We'll be looking at kind
of the intricacies of that,
how it's regulated
and other aspects.
And then we'll look
at incorporation
of unnatural amino
acids into proteins.
This is an important frontier.
This is an important
frontier in chemical biology
because it allows us to expand
the palate of what's available
for doing experiments
involving proteins.
And proteins do a lot, but they
only have 20 functionalities
available to them.
And in recent years,
chemical biologists
like Peter Schultz have been
inventing ways of expanding
that palate to go beyond
the naturally occurring 20.
We'll talk a little bit
more about that in a moment.
And then finally,
we'll end today
by talking about RNA libraries.
OK. Next week we'll
be on to week 6,
we'll be on to chapter
5, protein structure.
And again, there'll be two
lectures on protein structure
and then we'll be on to
chapter 6, protein function,
and again there'll be
two lectures on that
and we'll just keep
rolling them up.
OK? So, any questions
about where we're going
and things like that?
All right.
OK, some announcements.
I think I already
went over these.
I don't have to go
over them again.
I have office hours today.
I encourage you to come by.
Alternatively come by to
the TA's office hours.
My office hour next week
will be on Wednesday.
I believe it's 2:45 to 3:45.
OK. I already talked about
letters of recommendation.
Some last minute
announcements on the book
on the journal article report.
This is going to be due next
Thursday, a week from today
at 11 a.m. It is essential that
you submit both a hard copy
to me and also an
electronic version
through the turnitin.com
website.
Along those lines, it's not
really turned in officially
until the hard copy is
received and electronic version.
I will not accept any
email submissions.
OK, there's 120 of
you and I don't want
to get 120 PDF stuff
to print out.
OK. So, no email submissions.
It must be received
as a hard copy.
OK. So, very briefly, let me
review with you the requirements
of the article choice.
It's a good chance for
you to think and make sure
that you're following
directions.
Only research articles.
You know it's a research article
if it has a method section,
if it has some experiments
described in it,
and an experimental section
that discusses how the
experiments were done.
Now sometimes, those
experimental sections are found
in the supplementary
material that's online
to accompany the paper.
So, nowadays when
papers are published,
typically it's published,
there's kind
of an abridged version and then
there's a second supplement
that's also published
online concurrently.
And that supplement
includes a lot of details
that are too voluminous
to fit in the paper.
OK. Journals have a requirement
that you can't exceed a certain
number of words and it can--
include this certain
number of figures.
But there doesn't seem to be any
requirements on the supplement,
so what people typically do
nowadays is have these monster
size supplements.
So, last year for example
I published a paper
that was four pages
long and then it had
like a 25-page supplement,
single spaced, you know,
25 pages with like an
additional 15 figures
or something like that.
So, that's not all that unusual.
And so, if you can find in
that supplement the materials
and methods or experimental
or if you could find it
in actual journal article, then
you know that you are looking
at a research article, not
a review or news and views.
OK. And then again,
here're the journals
that we're going to
be using for this.
One thing I need to
caution you about is
that this is the Nature--
Nature the magazine
or Nature the journal,
not Nature pharmaceutical
reports.
OK? There's probably 25 journals
that have the title
Nature in them.
Only two of those are
acceptable for this project.
One of them is Nature and
the other one is Nature
Chemical Biology.
All of the other variants on
Nature will not be acceptable.
OK? So, Macmillan, which is
the publisher of Nature, again,
has a large number of journals
and they might say
slash Nature on them.
But unless they were
actually published in Nature
or Nature Chemical Biology,
they're not acceptable
for this project.
OK? And again, if
you give me something
and you didn't follow
directions, I'm just going
to hand it back to you
ungraded and tell you to redo it
and give you a late grade
for that assignment.
So, it's essential that you get
the journal article correct.
Yeah, question over here.
>> This question is kind
of for the whole class.
>> Yeah.
>> I tried to enroll
in turnitin.com.
>> Yeah.
>> And it didn't work
and I'm just wondering
if it worked for anyone else.
>> Did anyone have
trouble with turnitin.com?
You had trouble also?
Oh, so everyone had trouble.
>> Yeah, like--
>> Did anyone do
it successfully?
No. All right.
Thanks for pointing out for me.
I will have to take
a look at that.
Mariam, can you make a note?
>> Yes.
>> OK, thanks.
Thanks for letting me know.
That's good to know.
Last two points.
Must have been published
in the last year.
It needs to have the
number 2012 or 2013 on it,
and it must clearly focus
upon chemical biology.
So, it has to be a
chemical biology article
in the definition of chemical
biology that we're using
for this class which
all of you know, OK.
Thanks for pointing
it out about Turnitin.
Any other issues
that are coming up?
An issue that came up in my
office hours is how do you find
a journal article that's
relevant to your interest?
I'm hoping you all
know about PubMed.
There's ways of restricting
PubMed searches
to specific journals and I
encourage you to use them, OK?
Now, if your interests
are exceedingly obscure
like you're only really
interested in, I don't know,
dermatology, it's possible
there were no chemical biology
articles that covered, you know,
epidermal cells in
the last year, OK.
So, it's possible that you won't
find any chemical biology stuff
going on in that field.
In which case, you might
want to pick another topic
and expand your interest, OK?
But if your interest are
like HIV or something,
there are probably a dozen
chemical biology relevant
articles published
in HIV last year, OK,
maybe even more,
I don't even know.
OK. So, it's possible
you might have to change
around your topic a little
bit to suit what's available.
And again, I highly encourage
you to choose a topic
for this assignment
that will then lead you
into your proposal, right?
That way then you're reading
a state of the art paper
and when it comes time for
you to propose something,
you can basically take what
was in the paper, apply that
and go one step beyond, OK.
That's a really good
way to be creative.
Read something that's really
cool, get inspired by it,
bring in some new technique
or something like that.
And then before you know
it, you're on your own, OK.
Make sense?
OK. Any other questions
about the assignment,
anything like that?
OK. I want to talk
to you very briefly
about scientific writing.
As we've already discussed, this
is a major portion of the grade
and it's really essential
for your future career.
I believe very passionately
in the ability that--
of the importance of
effective writing.
So, I want to give
you a few guidelines.
These aren't hard and fast
rules, rather they're guidelines
that if you follow, I
guarantee it to you,
your writing will
be significant--
substantially better than just
everybody else's writing, OK?
So, the first of these
is strive for simple,
direct, clear sentences.
Think of your job as being
like a journalist, a reporter.
You want to have like a
Hemingway-esque style,
meaning really short
declarative sentences
where each sentence is clear.
At this point, your goal is
to make your writing as clear
as possible, OK, the
absolute clear as possible.
And the best way to do that
is have short sentences.
If your sentence goes
past about a line
and a half, it's
simply too long.
OK. There's a good chance
that the reader who is going
to be reading these things
very quickly, right--
that's the way everyone
reads nowadays--
will probably not have a
chance to keep track of it.
And so, that should clue
you in that it's time
to break the sentence
up into something short,
and every sentence needs to
have a subject and a verb.
And if you're choosing a verb,
choose one that involves
an active voice,
use the active voice.
If you don't know what
active voice means,
please go see someone on campus
who can help you with writing.
There's a writing coordinator
who can help you with that.
If this business about active
voice is totally mystifying
to you, get it checked out, OK,
you need to know
what that means.
Also, along those lines, if
the earlier thing I mentioned
about PubMed doesn't make sense
to you, go see the librarian
in the science library, OK?
There's people who are expert
at doing searches for the kind
of thing that you're doing.
OK. So, you know, whatever
I'm telling you to do,
if what I'm telling you
is totally foreign to you
and totally unfamiliar, then
it's incumbent upon you to seek
out resources that will
help you with this, OK?
And I can help you a little
bit during office hours,
but there's people who are even
better than I am at writing
on campus and even better
than I am at doing searches.
And you should seek them out
and use their expertise as well.
Double check your explanations
for understandability
or comprehensibility.
This is really important.
You should be able to take
your journal article report
after it's written
and then hand it
to the person sitting
to the right of you.
And that person should
be able to understand it.
So, make sure that
it's understandable,
that's really the true test,
that's one of the things I'm
looking for in good writing.
I should be able to
understand what's written, OK.
And then, this is
really important as well.
Avoid pronouns that are unclear.
This happens a lot
in this assignment.
It's very important that you
specify precisely the objects
and subjects of your
sentences, OK.
So, by pronouns I mean things--
I mean words like they, it,
you know, things
like them, these.
Those types of words
are inherently unclear.
So what happens is you'll have
some sentence like, you know,
bile acid drives up
production of immune cells
or something like that.
And then the next
sentence it will say,
these are terrible effects.
And what I don't know
is whether these refers
to the immune cells
or the bile acids.
It's just not clear to me.
And I know what you're thinking,
I know you're thinking, "Oh,
if he spend a little bit more
time on it, it will be clear."
But that's not the way
you want to communicate.
You want to communicate
so that the reader has one
and only one interpretation
of your writing.
And again, if you avoid pronouns
where it's unclear exactly
what's being referred to,
you can make your
writing much more precise.
And that's one of
the things you strive
for in good science writing, OK?
Questions about science
writing, about style?
This is the style that
I want you to follow
when you turn this in.
And this is how I'll be thinking
about it when I assign grades
to the written section
of your report.
OK? Questions about the style?
All right.
I want to talk to you
finally about plagiarism.
Again, this is one
of those things
that drives me crazy every year
no matter how many times I talk
about it.
This will be the last
time I discuss it though.
And the reason I'm
going to discuss it
with you now is I'm aware
that not everyone knows
what plagiarism is.
Or certainly everyone that gets
caught doing plagiarism claims
that they don't know.
So, we're going to talk about
it and define it very precisely.
OK. So plagiarism is
borrowing someone else's words.
And a relevant question is
how many words do you have
to borrow before it
counts as plagiarism?
OK. So, in science writing,
obviously you're going
to be borrowing some words,
OK, because you're going
to be discussing the
same sort of thing.
But what I'm interested
in is your own thinking
about those words, OK.
So for example, if you're
writing about Abl kinase
or this Abl protein,
then I'm expecting you
to borrow that word Abl.
It's unavoidable.
You can't get around it
without borrowing that.
But what I'm interested in
is how you think about Abl,
your own thoughts
about this protein,
and your own spin on this.
OK. So for example,
if a particular clever
sentence is something like,
although compounds
that are effective
in vitro proved are
proved to be too cytotoxic
for cellular assays,
the reported inhibitors
provide a proof-of-concept
for the efficacy
of disrupting Abl.
OK. So that's the example that
you found in literature and you,
you agree with this, this makes
sense to you, and you want
to have a sentence like
this in your own report.
Let's talk a little bit
about what plagiarism would
be if you borrowed this.
OK. So, what happens is
what students will avoid
or what students will attempt
to do is they'll go through
and they'll do a map to map
version of the same sentence
up here but in their own report.
And this is what
I call plagiarism.
So for example, they replaced
compounds with small molecules
and they'll replace effective
with acceptable activity.
And they'll say-- and instead
of in vitro they'll say
outside cells, too cytotoxic,
proved toxic, cellular
assays, in vivo,
the reported inhibitors,
the reported molecules,
demonstrate proof-of-concept,
efficacy of inhibit--
disrupting, inhibiting.
OK. To me, that's plagiarism.
OK. You've basically stolen
someone else's thought.
OK. Now, admittedly, you
have used different words.
You've done a one to one
mapping of different words.
But you haven't told
me anything new.
And I don't care about
someone else's thoughts.
I care about your thoughts.
The goal of this
assignment is for me
to learn about your thoughts.
OK. And the reason why I'm
telling you this is not
that I don't know that
the whole world is all
about ripping off
stuff off the web
and you're now putting a new
name on it and stuff like that.
That doesn't bother me.
That's not my concern here.
My concern here is that I
learn how creative you are
and how effective you
are at reading something
and then interpreting
it in a new way in a way
that hasn't been
interpreted before.
That's the goal of
this assignment.
So the goal of the
assignment is not
to simply recapitulate
someone else's ideas.
The goal of the assignment
is for you
to tell me your own ideas.
And that's what I want to grade.
I want to grade you
and not someone else.
And so that's why I
care about plagiarism.
OK. So let me show
you how to do this
so that you avoid plagiarism.
OK. So, this one down
here, this would be OK.
OK. So, what you do is you take
this sentence and then you think
about it a little bit.
OK. And you start to say,
"Well, you know what,
the problem here is first
the sentence is kludgy.
It's a mess.
It violates the rule about
too long a sentence, right?
It's complicated.
Short declarative
sentences are better.
So you're going to break it up.
You're going to say,
"The compounds reported
in this paper were too
toxic for cell studies."
That's unavoidable.
OK. Right, this is a fact.
There is no way that
you're going
to escape not being
able to state the facts.
You can put the facts
in your report.
In fact you need to.
It's the second part
that interests me more
which is the interpretation.
And what it said here
is, "The report, however,
advances cancer therapy
by describing a novel mode
of small molecule
inhibition-- disruption of Abl."
OK. So what you've done here
is you've put the report--
this report, this scientific
discovery in the context
of the larger field
which is cancer research
and then that's your spin on it.
OK. That's what you've
done to help me know
about your creativity.
OK. And that's really where--
that's the value
added that I'm looking
for in a good scientific
communication.
OK. I know that you're going
to have to restate the facts.
You might even have
to restate some
of the experimental methods.
That doesn't bother me.
What I'm really interested
in though is how you
interpret those facts.
How you spin the facts.
How you put them in the
context of chemical biology
and in the field and
in cancer research.
That's the part where
you get the A grade.
OK, that's the part
that interests me.
OK. That's the part
that I can say, "Oh wow,
this person is thinking
in a unique way."
That's the part really I'm
looking for in this assignment."
OK, does that make sense?
OK. And I'm not trying to scare
you about this plagiarism stuff,
but it is scary because later
in your career you can get fired
from your job for even
small amounts of plagiarism.
The great historian, Doris
Kearns Goodwin was caught out,
you know, borrowing something
like half a sentence.
Half of a sentence was enough
to tarnish a lifelong of work
where she had achieved so much.
And don't let that
happen to you.
OK. That's not-- it's not--
it's not fair for all
of your hard efforts.
OK. So you do not want
to be in that position.
And so now would be
a time to resolve not
to let that happen, OK?
Any other thoughts or
questions about plagiarism?
Does this make sense?
I'm not giving you a definition.
I'm giving you an example.
Hopefully the example
makes total sense.
If it doesn't, ask now.
OK, so again, I will
be searching for--
I will have the TAs actually
doing Google searches
and searching for this.
It's very easy for us to spot.
And if we do, we do come down
very heavy on this very hard
because this is an
academic integrity issue.
We will report people
to the dean.
There will be serious
consequences.
I don't want it to
happen and so if we manage
to have a whole year where
we have two assignments
with zero plagiarism, then
I'm going to bump the grades
up that are on the
interface between A's
and B's, and B's and C's.
OK. So that will happen
for the whole class.
So there's the stick.
The stick is the dean's office.
And there's a carrot.
The carrot are higher grades.
Help me get to the carrot side.
I will tell you, I've been
offering the carrot now
for many years and I've never
ever been able to deliver it.
This could be the year.
OK. I know, it depresses me.
That's why I keep
talking about this stuff,
because every time we have
someone in my office like, "Oh,
I did think that
was plagiarism."
Well, now you know.
All right, any questions
about this concept?
OK. Oh yeah.
[ Inaudible Question ]
Oh, I'm so, so glad
you asked that.
OK. So, this is brilliant.
OK. So, the question
I got was what
if you use this first
sentence and then right
after that you put a number
two and that's the reference
down to the paper that
this was-- this came from.
The answer is no, that
would not be acceptable
because you'd still be claiming
that these words
are your own words.
OK. The way that would be
acceptable to use this would be
to use the first sentence,
the published sentences,
put it in quotation marks.
Quotation marks designate
that you borrowed it
from someone else.
And then, put the reference
back down to the citation.
OK. That's really,
really important.
Everyone who plagiarizes
includes references.
Not everyone, but 90 percent
of the people put references
to the stuff that
they're plagiarizing from.
OK. And it doesn't count.
That still counts as plagiarism.
Even if you reference
the stuff that--
the source that you
borrowed the stuff from, so.
Let's see.
Are you here just visiting or
are you here for the class?
>> For the class.
>> All right, welcome.
Here, why don't you have a seat?
So, that way then you
will be comfortable.
So, I just don't
want you to look
so uncomfortable
for the whole class.
>> No, no.
Don't worry.
>> So, OK.
>> Don't worry about it.
>> Well, I do worry about it.
Have a seat here or here, so.
OK, you get the hot seat.
OK. Any questions about
any of the announcements?
Any questions?
That was a good question.
All right.
Let's move on.
Here's what we saw last time.
What we saw last time is RNA
is this malleable polymer
that folds upon itself
as it forms Watson-Crick
and Hoogsteen base pairs.
And this malleability is a
really fantastic property
because it gives
these biopolymer lots
of different shapes to allow it
to access different structures.
And these structures
as we're going
to see today confer function.
OK. So, one of the themes of
the class is that structures
of biopolymers leads
to their function.
Form follows function
in biology.
Not always but most of the time.
We talked a little bit
about different base pairs.
I also wanted to emphasize
that the molecules
we're talking about,
the transcription
factors, the enzymes, RNAs,
these are dynamic molecules.
These are molecules
that live and breathe,
that have motions associated
with them, that have kinetic
and dynamic parameters
associated with them.
One of the dangers of
teaching a class like this is
that I show you a bunch of
pictures of beautiful molecules.
OK. It's like going to
the zoo or something.
But instead of being at
the zoo, you're at a zoo
where everything
is frozen in place.
And you know that's not
really the way animals exist.
You know animals
like to move around.
They like to be roaming around
the savanna or their cages
or whatever it is
that they're doing.
Biomolecules similarly move
around, they have dynamics.
And when I talk about something
like a transcription factor
and I describe it
riding the rails
of the phosphodiester
backbone, I really mean it.
That is exactly what it's doing.
It is cruising on that
DNA pi-way as it looks
for the correct base
pairs to grab on to.
This is as essential.
You must start thinking
about these molecules
as having a fourth dimension,
of having motions associated
with them, and this is one
of the frontiers in
chemical biology.
And it's an area that we need
to continue to push and explore
and understand better.
Because in doing so, we're
getting a much richer view
of how things are
happening inside cells.
OK. I'll try to continue
to emphasize this point.
We talked a little bit about how
transcription factors scan DNA
sequences at very high speeds.
And then, they form distinctly
different interactions upon
finding the specific sequence
that they want to bind.
In other words, they're zooming
along these phosphodiester rails
and when they find that
particular correct structure,
they kind of scrunch down
and they form interactions
either directly
with the DNA bases or indirectly
through water molecules
with the DNA bases.
And that's what allows them to
bind to a particular sequence
of DNA, recruit the other
factors that are required
for transcription,
and eventually recruit
RNA polymerase
and kick off transcription.
At the end of Tuesday's
lecture, we introduced you
to this yeast two hybrid screen.
This is a very powerful
tool that allows us
to test protein-protein
interactions in cells.
It's used pretty ubiquitously.
I would say last few years its
use has fallen off a little bit.
But it's still one of the major
tools that are used in, say,
biochemistry, molecular
biology laboratories,
and even chemical
biology laboratories.
I told you about the variant
that had two binding partners.
There are, however, variants
that have three binding partners
where you can have, say,
two proteins that are kind
of like the bread in a sandwich
and then a small molecule
in the middle that's kind of
like the meat in the sandwich.
And the three of
these things have
to come together before the
transcription takes place.
And then it's also
possible to look for things
that push apart the interaction
if you're turning
on say a toxic gene.
And so that's called
the reverse two hybrid.
And so there is a,
you know, half a dozen
or so different variants of
this yeast two hybrid available,
this yeast hybrid idea
that are available and--
but they're all based upon the
idea that you could separate
out the activation domain
from the DNA binding domain.
And in doing so, you
end up with something
that then it could
be recapitulated,
that can be reformed
upon an interaction,
upon formation of
an interaction.
OK. Any questions about
what we saw on Tuesday?
Questions about anything
like that?
All right.
Well, I want to move on then.
I want to talk next
about translation
and actually I think I have
just a little bit more to talk
about in terms of
transcription and messenger RNA
and then we're on
to translation.
OK. So, let me get to where
we left off last time.
OK. So, last time, I ended with
the observation that bacteria
and eukaryotes have very
different levels of complexity
in terms of their mRNA
processing, right?
Where bacteria have
DNA that's transcribed
and then the same RNA leads
directly to translation.
Whereas, eukaryotic cells have
DNA that's transcribed and then,
introns or inserts are cut
out, the exons are rejoined
and then the mRNA is modified.
At one end there is a
polyA tail that's added.
At the other end, there's a cap.
And all these must take
place before translation can
actually happen.
So, why don't we dive
right in and take a look
at it a little bit
closer at the chemistry
of eukaryotic gene translation--
or sorry, the chemistry of mRNA
processing before translation.
OK. So, here's, you know,
here's a little short--
let me just get some water.
Sorry. Throat is very dry.
Short summary of what
the changes look like.
OK. So again, DNA
leads transcription.
You get this RNA transcript.
The RNA transcript is first
kept at the 5 prime end
and there is this G,
methyl-G cap that's added.
And this is kind of a
weird looking thing, right?
It has a triphosphoester,
diester backbone.
And it has some weird linking.
This is 5 prime to 5 prime.
And then you have
this cap over here.
But this evolved in a
way to allow the mRNA
to be shuttled very
quickly to the ribosome.
OK. We'll talk a little
bit more about how
that works in a moment.
At the other end, the 3 prime
end, the messenger RNA is tagged
with a long sequence of A's.
So this is called a polyA
tail at the 3 prime end.
And then finally, the
introns are spliced out.
They're actually chopped out.
They're either chopped out by an
active process involving other
proteins or sometimes
just spontaneously.
And then finally,
the leftover stuff,
the exons are actually
expressed as protein.
OK? So there's a lot of
modification that takes place
after the messenger
RNA is synthesized.
And why don't we take
a closer look at this.
Let's start with this GTP cap.
This is the triphosphate.
There is the triphosphate
over here.
This is a weird looking
sequence.
Notice the extra methyls.
There's one here,
there's one here.
Other than that, it looks kind
of like a G. This has
the function of helping
to load the 5 prime end of the
messenger RNA onto the ribosome.
OK. So it gets things going.
The way this bond is
formed for the methylation
of that is distinct
from I'd say 99 percent
of carbon-carbon bond
forming reactions in biology.
But it's also number two
in terms of its importance,
so for that reason we
should take a moment
to talk about this.
First, let me just
digress for a moment.
We'll talk later about
how 90 percent plus
of carbon-carbon bonds
are formed in biology.
They're formed using
an aldol reaction.
This actually is a rare example
of a carbon-carbon
bond or-- oh, sorry.
This is actually not
a carbon-carbon bond.
This is a carbon
heteroatom bond.
This is a rare example though
forming a bond to carbon not
through an aldol reaction.
OK. This is actually
used as an SN2 reaction.
And it's a straightforward
nucleophilic attack
by the lone pair
on this nitrogen.
Notice that this lone
pair is not involved, it--
not involved in aromaticity, so
it is a very good nucleophile
to attack the methyl group
of this S-adenosylmethionine.
S-adenosylmethionine has a role
of delivering methyl groups.
OK.
And actually, now that I
think about it, this is--
that phrase up there is
not so helpful to us.
OK. So, apologies.
All right.
Now, on-- that's on the 5
prime end of the messenger RNA.
On the 3 prime end
there is an appendix,
a series of A's that
are appended.
And the exact number varies
but it can be really long.
It goes between 50 to
200 bases of just polyA
that are simply stuck on there.
And I know what you're thinking.
You're thinking this is a total
waste of energy for the cell.
Why would it bother doing this?
OK, what is-- what
is up with that.
And this is useful
because it binds
to a polyA binding protein.
There's a protein shown
here that evolved to bind
to this polyA's and that
helps direct the mRNAs
to the ribosome.
OK. So it turns out that's
actually a useful thing.
So these two ends of
the messenger RNA act
as specialized handles where
they have a directionality.
And directionality as
you know matters a lot
in the sequences of
RNA or DNA, right?
There's only one direction that
leads to a correct sequence.
Other direction leads
to gibberish.
Now, because all of the
messenger RNAs are appended
with this polyA tail, there
is a really effective way
that we can use to isolate
all of the messenger RNAs
in the cell and throw
away everything else.
So what you can do is you
could set up a solid support
that has a bunch
of T's bound to it.
OK. And then, hybridize
that to all the stuff found
in the cells.
The only things that will
stick are the messenger RNAs
which have a polyA tail.
OK, so in practice the way this
works is we use the carbodiimide
DCC which we previously saw
forming amide bonds back
in Chem 51.
But here, we're going to use it
to form phosphodiester bonds.
And what you do is you
simply add an excess
of T deoxy-- or sorry.
This is T monophosphate
and with this DCC.
And then, in the presence of
cellulose, this will react
with the cellulose, the primary
hydroxyl of the cellulose.
Notice that this is cellulose.
Cellulose of course is beta
D glucose that's polymerized.
And here's the primary 6
hydroxyl of the glucoses
and that will react
with one of these T's.
And then the T's will polymerize
with each other in the presence
of this coupling agent, DCC.
Mechanism here is
exactly like what we saw
when we saw formation of
amide bonds using DCC back
in sophomore organic
chemistry, back in Chem 51.
And if that mechanism is not
apparent to you, please go back
to your sophomore
organic chemistry textbook
and look it up again.
OK, relearn that mechanism.
That's a useful one.
OK. At any case, what you end
up with then is basically paper
that has a bunch of T's
covalently linked to it,
a polyT just sequence, just kind
of hanging out there in space.
And you then solubilize this.
You dunk it in water
and you flow
over this the extracts
from the cell.
So almost everything in the cell
washes past the paper except
for the messenger RNAs because
the messenger RNAs are now going
to form Watson-Crick
base pairing A's to T's.
So you have polyA on
the messenger RNA,
polyT on the cellulose and
the two of these hybridize
to each other and that
allows you to isolate all
of the messenger RNAs and wash
away all the stuff that's found
in the cell.
Make sense?
OK, so this is very
routinely used
but I don't think most people
spend too much time thinking
about how this is synthesized.
It's pretty straightforward.
OK. Let's talk about
the next step
in the processing
of messenger RNAs.
So after-- after they're capped
on one end with the GTP cap
and on the other
end with the polyA,
the introns have
to be spliced out.
There is a bunch of
snRNPs, short nucleotide--
ribonucleotide repeats
that are--
that sort of pink pile on the
introns, bring stuff together
and set
up a transphosphorylation
reaction, OK.
This is where you get a transfer
of a phosphodiester
bond from here to here.
So, it's just a simple
exchange and that has the effect
of cutting out the intron
in this interesting
[inaudible] structure.
Details here not so
important for us, OK.
This does, however, bring up the
really interesting observation
that RNA is capable of
catalyzing reactions,
and this is kind of our
first example of this
that we're looking
at at some detail.
So, I want to show you a more
canonical example of RNA acting
as a catalyst, and that
example is the classic
hammerhead ribozyme.
OK. So, here is the structure
of the hammerhead
ribozyme in green.
This is a naturally
occurring RNA sequence,
and in red this is a sequence of
RNA that's targeted for cleavage
by this hammerhead ribozyme.
And what the hammerhead ribozyme
does is it orients a base close
by to the 2 prime
hydroxyl to deprotonate
that 2 prime hydroxyl.
There's also a magnesium
bound and that sets
up a nucleophilic attack on the
phosphorus of the phosphate.
This is starting to look
really familiar, right?
We've seen ways to cleave
RNA before and you know what,
this is identical to it.
The only difference here is
that the polymer organizing this
or catalyzing really this
attack happens to be in RNA.
And so, whenever
we see a catalyst
that is an RNA that's catalyzing
some reaction, we're just going
to call it a ribozyme, OK.
So it's like an enzyme except
it's made out of RNA, OK.
Recently my colleague
Andrej Luptak discovered
that these cells--
that these RNA--
or these ribozymes are
very widely dispersed
across all creatures
found on the planet.
He's found them in humans,
he's found them in starfish,
and a whole series
of other organisms.
Again, all of these
require magnesium.
Magnesium is playing this
key role as a Lewis acid.
It's stabilizing the negative
charge that's surrounding this
phosphorus and making it
a better electrophile.
OK, so, in the cell, the
cell has a messenger RNA
and then it has to
eventually degrade it.
So the cell-- and furthermore,
the cell is, you know,
constantly coming up with
stuff that, you know,
that's getting [inaudible] with,
say, viral RNA, so there has
to be a mechanism of destroying
RNA after it's finished, OK?
So, after its time has come,
after the translation has taken
place, there needs to be a way
of degrading the messenger RNA.
And for that matter,
it's useful to be able
to degrade RNA that's coming
in from, I don't know,
viruses and things like that.
So, this has been taken--
OK, so, the way this works,
one way to target messenger
RNAs for destruction is
to use an anti-sense DNA.
So, the anti-sense
DNA will recruit--
well, after it hybridizes
to the messenger RNA,
it will recruit ribonuclease H
and this will then
destroy the RNA, OK?
So, this idea of using
anti-sense DNA as a way
of targeting specific
messages sent
out by the cell would be
amazingly powerful, right?
We'd have a way, say,
of shutting down cancer
if we can target
specific messenger RNAs
that are associated with cancer.
This would be very,
very powerful, OK.
So, in recent years, there
is-- or not recent years,
has been going on
for like 20 years.
There's been attempts to
develop anti-sense therapies.
These are therapeutics that will
do something exactly like this.
They'll deliver a
sequence that hybridizes
to specific messenger RNAs and
then recruits ribonuclease H
to degrade that message
and prevent it.
OK. This is distinct from
conventional pharmaceuticals
which often feature
a small molecule
that inhibits some enzyme, OK.
So, the standard way
to do this would be
to allow the messenger RNA
to be translated resulting
in an enzyme and
then destroy the--
or not destroy it but
disrupt the enzyme
by inhibiting it using some
small molecule inhibitor.
OK. We saw examples
of this, right.
We saw for example
chloramphenicol targeting
chloramphenicol
acetyltransferase, right?
And so, in this case, instead
of like targeting the enzyme
that results from translation,
we're going to kill the message
itself, prevent translation
and in this way prevent
this enzyme
from doing its function, OK?
So, it's a really distinct
mode of therapeutics and it's--
I would say a couple of
years ago up until, say, two,
three years ago, I was deeply
skeptical about the whole thing,
but there's been
recent progress.
I believe there's now two
drugs that have been approved
by the FDA based
on this principle
and things are starting to
look a lot stronger, OK.
Here is what the problem was.
Here is why this took so long.
OK. So, here is one example
of a FDA approved drug
that uses this principle.
The drug is called
formivirsen and it targets CMV,
cytomegalovirus RNA
and it does this by--
so here's the formivirsen.
It forms perfect Watson-Crick
base pairing with the CMV RNA
and that in turn recruits
RNase H which are the scissors
to chop apart this sequence, OK.
Now, the real problem here
is that these sequences,
the anti-sense sequences--
notice that they're called
anti-sense because they have
to have the Watson-Crick
base pairs.
So, instead of having it-- and
so, there has to be C's and G's
and A's and T's lining up,
although I'm looking now
and it doesn't look so neat
from this illustration.
But you know what I mean, right?
So, here's T's and A's
and G's and C's lining up.
So, that's why they're
called anti-sense.
But a major challenge is
delivering in these biopolymers
in a way that they can
actually get inside the cell
and be effective.
Challenge number
one is that the DNA
and RNA are pretty short
lived outside the cell.
We've already discussed RNases.
There's plenty of RNases
that are circulating.
There's also plenty of DNases.
Those tend to chop apart
wayward strands or DNA or RNA
that happen to be
floating around, OK.
So, what people have been doing
is modifying the backbone.
So, instead of a
phosphodiester backbone
of this anti-sense therapeutic,
instead one of the oxygens has
been replaced with the sulfur.
And that backbone modification
prevents the degradation
of the targeted sequence.
OK. So, that's one
thing that's happening.
Here are some examples of
other backbone modifications.
In one, the phosphorus
is replaced entirely
with amide bonds in a
peptide nucleic acid
and perhaps the most
effective examples
of these are these
morpholino oligonucleotides
that have this weird
morpholine type backbone.
These tend to work
really well, OK.
These morpholino
oligonucleotides are used
routinely in chemical biology
in biology laboratories as a way
of knocking out specific
messages.
So, you can take some
mRNA, take that sequence,
convert it to an
anti-sense and then order
up a morpholino oligonucleotide
which incidentally is not cheap
but it can be done and you
can then use this directly
in your experiments.
Notice that the big change here
is a change from having lots
of negative charge
on the backbone
to having neutral backbones, OK?
That helps quite a bit in terms
of delivering the therapeutic
inside the cell, right?
Negatively charged things
have trouble passing
through the phospholipid
membrane layer
that surround cells, right?
We talked about how that's--
it has an outside that's polar
and an inside that's
hydrophobic.
Charged things don't
like fitting
through that hydrophobic region
of the phospholipid
plasma membrane.
And so for this reason,
these neutral things
are more effective.
[ Inaudible Remark ]
Yeah.
[ Inaudible Question ]
Oh yeah, OK.
Yeah, it means it
doesn't happen anymore.
There really should just
be a big X here, OK?
Thanks for asking, Anthony.
All right, let me show you--
So I've said before this is
useful in the laboratory.
I want to show you an
example of this, OK.
So, what we're doing
here is we're interested
in targeting a particular
messenger RNA
that encodes this
vimentin gene, OK.
And if the vimentin
protein is produced,
it will be stained
using an antibody
and the antibody happens
to be dyed in red.
So you will see it under
fluorescence micrograph image.
And I think I'm going to turn
down the lights even more
because this is a
little hard to see
but it looks a little
bit better on my--
so, I'm just going to turn
this off very briefly.
So, here's cells in blue.
This is the nucleus
being stained
with the fluorophore DAPI.
It happens to bind well to DNA.
I think we might have even seen
a little bit about it earlier,
seen structure earlier.
And again, in red, this is
the vimentin protein, OK.
So, this is basically
the negative control.
This is short interfering RNA
that does not target
the vimentin gene
and it's really essential that
you do these controls, OK.
So you've treated
these cells with RNA,
but in this case it's RNA
that doesn't have the
anti-sense necessary
to target the message
encoding vimentin.
OK. Now, over here,
here are cells
that have been targeted
using this siRNA
which again is this RNA
interference mechanism
that we've been talking about.
But know, the anti-sense targets
the mRNA that encodes vimentin.
And notice that there's
very little red.
There might be a little bit
here, but for the most part,
it's totally clear, the red,
yet you can still see the
nuclei of the cells, right?
You can still see these blue
nuclei, which is the DNA
and the nuclei being
stained, OK.
Can everyone see that?
OK, so this works
really well, all right.
And again, the way
this is going to work
in this case is you
have a plasmid
that encodes this siRNA, OK.
And furthermore, it's even
more complicated than that.
What you do is you actually set
up-- so, you have this plasmid,
remember recall that
plasmids are circular DNA.
The plasmid encodes the
sequence that's going
to be the anti-sense
sequence and in practice,
this actually encodes not
something that's simply an
anti-sense sequence, rather
it encodes both the sense
and the anti-sense
sequence into a hairpin, OK?
So, over here, this upper
strand is the sequence--
the mRNA, it looks
kind of like the mRNA
that encodes the vimentin gene,
but it's just a little
fragment of that.
And then, there's a little
hairpin, right, that's a loop
that we've seen before and then
down here on the lower strand,
this is the anti-sense sequence.
So, sense, anti-sense.
OK. So, now what happens is this
short hairpin is now a section
of double stranded RNA and
that activates a mechanism
in the cell called dicer, OK.
And dicer goes through
and systematically looks
for anti-sense strands
of messenger RNA
that have the sequence and
catalytically goes through
and starts chopping
those apart, OK?
And it chops one after
another apart, OK.
And if you want to learn more
about dicer and Argonaute
and the other proteins
involved, you can read
about it in the text, OK.
All right, let's switch gears.
I want to-- any questions
about mRNA processing?
Questions about that topic?
OK, it turns out it's a really
active area of research.
It's always been active,
it's always fascinating.
There's new surprises that
are constantly coming along.
I want to switch gears though, I
want to talk to you a little bit
about what happens next.
The messenger RNAs are
eventually delivered
to the ribosome.
In prokaryotes, the
ribosome binding site
or RBS is something called
the Shine-Dalgarno sequence.
It's more of a guideline
than a sequence.
You can actually get
away with some variations
on this Shine-Dalgarno sequence.
But it turns out that
if you don't program it
in, nothing happens, OK.
And every so often, you know,
someone new joins the
laboratory and designs.
There are protein-- you
know, there are construct
to be expressed and
nothing happens.
The cells refuse
to take it up it is
because they've forgotten
the Shine-Dalgarno sequence.
So, it is essential.
In eukaryotes, there's something
called the Kozak sequence
and this idea is the same.
There's an area where the
messenger RNA is bound
by the ribosome and that kind
of gets everything going, OK.
Now, the actual ribosome
catalyzing amide bond formation
is a pretty straightforward
reaction, simply consist
of amines attacking esters.
OK. So, recall from
back in Chem 51
that if you mix together
amino acids and you boil them
for a long time, you
can form an amide bond.
But the efficiency was very
low and you don't have control
so much over which amide
bond was going to be made.
OK. So, we talked about
why it was important
to activate the carboxylate
of the amino acid
to form an amide bond with
greater specificity, right.
If you were in 51c with me,
we had this conversation.
And again, if this
conversation about activation
and DCC is totally foreign to
you, totally confusing, go back
and take a look in your textbook
from sophomore organic
chemistry, OK?
So, DCC I've alluded
to twice in this class
and both times I told you
if you don't know what it
is, go back and look, OK?
So, in this case, the cell
doesn't have access to DCC, OK.
Instead its activation agent
is forming the amino acid
into an activated ester, OK.
So, here's an amino acid.
It happens to be methionine.
And R over here is
the transfer RNA.
And so what's going
to be happen is the--
this will form an amide
bond with a N terminus
of a nascent peptide that
will attack this ester.
OK, so this will be
the first amide bond.
And the second one will be
the next amino acid delivered
to attack transfer RNA of
this threonine, et cetera.
OK. So the ribosome
is stringing together.
These transfer RNAs that have
activated amino acids attached
to them.
So I'm going to be referring
to these activated amino acids
as aminoacyl-tRNAs where
acyl refers to the fact
that these are formed into
ester functionalities.
OK, make sense?
OK. And furthermore,
it makes sense
that we have this
activated ester.
Another way of thinking
about this is
that hydroxide is a
terrible leaving group.
And so instead of
having hydroxide
as a leaving group
we have alkoxide.
It happens to be a special
alkoxide in the catalyst,
et cetera but that's the idea.
OK. All right.
So, chemically, what's happening
here is the incoming amino acid
is attacking this ester.
OK. So this is a straight
attack of a carbonyl.
And then, you form this
tetrahedral intermediate.
The tetrahedral intermediate
then collapses
and the result is
formation of an amide bond.
And I have two possible
mechanisms here.
One that can take place under
basic conditions on the top
and one that takes
place under acidic,
more acidic conditions
on the bottom.
Either one of these
is legitimate.
OK. And both of them lead to
formation of an amide bond.
OK, straightforward mechanism.
It's one that I'm
hoping you're familiar
with from back in the day.
And if not, go home, try it
a couple times for your self.
It should be pretty
straightforward.
OK. So let's take a look
at the ribosome itself.
The ribosome is really
a mega machine.
It's a huge machine that has
upwards of 20 different parts
to it, constituent parts.
These include both proteins
which are shown here in blue,
and also RNA sequences that
are all kind of put together.
OK. So, here's the
messenger RNA being red.
And then here's the
peptide being spit back
out of the ribosome.
Notice that the action site,
the site of action
called the active site is
in the very center
of the ribosome.
And if you look at the center of
the ribosome, it's mainly RNA.
OK, so in fact, the
ribosome is a ribozyme.
It actually it relies upon RNA
to catalyze this
amino lysis mechanism
that I showed you earlier.
OK. Let me see if I
got everything on here.
OK. So, a mixture of RNAs
and proteins, et cetera.
OK. Here's a-- it turns out that
because it plays such a key role
in the cell for protein
translation,
it's also a major focal site
for antibiotics to target.
It's hard for antibiotic
resistance to emerge
with this one because it's
hard to mess up the ribosome
without losing its
catalytic efficiency.
It's just too important
for the cell
to start messing around with.
And so, many antibiotics
target the ribosome.
And one of these for example
is the antibiotic tetracycline.
Tetracycline binds directly in
the active site up here and also
as a lower affinity
binding site down here.
OK. And it's shown in purple.
OK. So, tetracycline routinely
given, anti-acne medication,
effective way of
killing off bacteria.
It happens to have
slightly higher affinity
for the bacterial ribosome
than the human ribosome
but the differences
are fairly subtle.
OK. So here is the structure
of tetracycline down here.
It has four rings,
hence the name.
And again, this targets
the ribosome.
There's a whole series
of different molecules
that target the ribosome, things
like kanamycin, erythromycin.
This is another one that should
be familiar to those of you
who have bacterial infections
at some point in your life.
You've probably encountered
erythromycin.
OK. It's a macrolide antibiotic.
We'll talk more about these
polyketide antibiotics
in a few weeks, probably
towards the end of the class.
But it also it targets
the ribosome.
Streptomycin also
targets the ribosome.
Totally different structure.
This is an amino
glycoside antibiotic.
And then, they are antibiotics
that target not the ribosome per
se but the machinery that helps
to load tRNAs up
onto the ribosome.
And there are two
ways of doing this.
One is targeting
EF-Tu shown here.
Kirromycin and another one is
targeting another protein called
EFG which is this one over here.
In any case, all five of
these molecules operate
by a common mechanism.
They all operate by shutting
down protein translation
for the cell.
OK. So this is one
of those areas
where it's just really
rich with lots and lots
of different antibiotics.
And we'll see this
time and again, right?
We talked about molecules
that target DNA.
We talked about molecules
that target the ribosome.
They're sort of like
Achilles heels for the cell,
areas that are real choke points
that antibiotics can get in
and mess up pretty readily and
do it in a broad spectrum way
without killing lots and lots
of different species really,
a bacteria in this case.
OK. So, let's talk
about translation.
So, translation starts
with a start codon.
In eukaryotes the start
codon encodes the amino
acid methionine.
So the N terminus of all of--
all proteins synthesize
the eukaryotes,
starts off with a
formyl methionine.
Notice that there is this
[inaudible] that's been appended
to it.
That's just another way
of getting things going.
And so-- oh sorry.
This is the bacteria case.
Bacteria starts with
the formyl methionine,
eukaryotes no formyl methionine.
Let's take a closer
look at the tRNA.
TRNAs again bring amino
acids to the ribosome
as activated esters,
as aminoacyl tRNAs, OK.
And at one end of the
ribosome-- oh, I'm sorry.
One end of the tRNA,
the 3 prime end,
the amino acid is
loaded in as an ester.
Way down here at the other end,
there's three bases called
the anticodon which will try
to hybridize the messenger RNA.
And if they hybridize
that tells the ribosome
that it's the correct sequence,
the correct amino acid
that's being loaded
in for amide bond formation.
This is really essential,
this base pairing
between anticodon
and codon loops.
This is what allows
the correct--
the synthesis of the
correct sequence.
Right, otherwise, you know,
you have your DNA up here,
your messenger RNA
and your proteins.
This last-- this is
the last step really
in the central dogma
of molecular biology.
This is what gives you
the correct sequence
that was encoded by the
DNA in the first place.
OK. Now, here's the
way this works.
So, the messenger RNA is read
out in three base pair
sequences called codons.
OK. Each one of three bases
leads to a different amino acid.
And I'm showing you
what the amino acids are
of the 20 amino acids on
this genetic code diagram.
OK. Now here's the way you
read this genetic code diagram.
You start in the center
and this tells us.
Let's just start with G. OK,
so if the first residue is G
and the second one is C,
and the third one is C,
so GCC would be to alanine.
OK. CAG leads to glutamine.
UGA, however, leads to stops.
OK, so there's two possible
stop codons-- sorry.
Three possible stop
codons that are useful.
Those tell the ribosome,
kick it off.
You know, kick off
the messenger RNA.
You're done.
OK. And that stops the sequence.
OK, so there are 64
possible combinations.
There's only 20 amino
acids plus some stops
so what this means then is
that several codons encode
for the same amino acid.
OK. And in practice there's
some slight preference
for some codons over others.
And this preference is
dictated by the levels of tRNA.
So there are some
tRNAs that are present
in higher concentrations
in the cell.
And in practice when you
design protein overexpression,
you look for codons
that are more popular
than the less popular one.
There are some codons that
are exceedingly rare inside
the cell.
And if you have a choice of
say four different codons,
in the case of [inaudible]
down here,
you'll choose the
most popular one.
I don't remember what it is
but you would choose let's
say ACC rather than ACU
because it's represented
more often in genome.
OK. So here's the way--
here's what it looks like.
DNA has a sense strand
and an anti-sense strand
during transcription.
A copy again is made
at the sense strand
and then this copy
is translated out.
The sequence up here results
in the amino acid protein
sequence down here.
So for example, ATG we've
seen is a start codon.
I didn't call it a start codon
but we know it encodes
methionine.
OK. ATG, OK, methionine, right.
OK. So this encodes methionine
and over here ATG as a codon
at the DNA level results
in methionine down here.
OK. Similarly, GGG,
GGG encodes valine,
and so over here
results in valine.
OK, and so you can do this
pretty readily if you have one
of these genetic code,
you know, wheel diagrams
that I'm provide--
that is in the book.
So you can very readily
figure out what a sequence
of protein will result.
OK, makes sense?
OK. Now, crucial step.
At some point, you have to
load the correct amino acid
onto the tRNA.
If the amino acid is mismatched
with the anti-codon down here,
the cell is in big
trouble, right.
This is essential to get
the correct sequence out.
And so, enzymologist debated
for a very long time how
the molecular recognition
of the tRNA would work with--
with recognizing just
three bases of the--
of the anticodon
loop of the tRNA.
And in practice, what we
found is actually the enzyme
responsible for this loading,
an enzyme called
aminoacyl tRNA synthetase,
this enzyme is a monster, OK.
So, it forms a dimmer.
It's shown here in green.
These are two tRNAs, one on the
left, one on the right side.
And notice how this
thing is just grabbing
on to both of these.
So, it's interacting not just
with the anti-codon down here
to read out the sequence
but with lots
of other places along the tRNA.
And then furthermore up
here, this is the active site
where the aminoacyl-- the
amino acid forms an ester bond
of this 3 prime hydroxyl.
I'll show you in a
moment what the mechanism
of that reaction is.
But again, notice that the
aminoacyl tRNA synthetase
engulfs the whole tRNA.
It's in a bear hug and so
there's more interactions
than just the anti-codon loop.
And furthermore earlier,
do you remember I told you
how tRNAs especially were very
heavily modified.
Back when we were looking at
say the clover leaf structure
of tRNA, I said how
heavily modified they are.
That heavy modification
helps direct the correct tRNA
over here to the correct
amino acid up here.
And it's being read
out by this protein,
by this enzyme that's
checking it over.
OK, makes sense?
All right, so, let's take a
closer look at the mechanism.
In practice, the mechanism
involves activating
the carboxylate.
Because again, carboxylates
are very inert,
they don't like to be--
they don't like to form
bonds all that ready.
Hydroxide is a bad
leaving group.
And so in practice,
this is activated
by forming an acyl phosphate
intermediate using ATP
as the activating agent.
OK. So, phosphate is kind
of like nature's
tosylate or mesylate.
It's some super leaving
group that's ubiquitous,
that's found all over
the place in biology.
And this is going to work
by forming a readily
hydrolyzable bond.
OK. So for example, the
glutamyl-tRNA synthetase starts
with glutamine-- or sorry,
glutamic acid, glutamate,
and activates this through an
acyl phosphate intermediate.
And then the glutamyl-tRNA
synthetase ask, OK,
is the acyl phosphate
intermediate
available glutamate.
And if it's not glutamate, then
it hydrolyzes this intermediate.
And if it is, then it
adds the amino acid
to the 3 prime hydroxyl
of the tRNA.
OK? So it's a little
bit complicated.
There's actually a couple of
steps where things are checked.
The tRNA is bound and it's
gripped in a big bear hug
where it's actually making
sure that has the correct tRNA,
making sure by testing
the anti-codon
but also looking along
the length of the tRNA.
And then, different
intermediates,
acyl phosphate intermediates are
brought up to the active site.
And the enzyme asks, is
this the correct one?
Is this glutamate?
And if it's glutamate
then it forms a bond.
And if not, then
it kicks it off.
And when it kicks it off
it actually hydrolyzes the
phosphate of the-- the
phosphate intermediate.
OK. Any questions so far?
Yeah, way in the back.
[ Inaudible Question ]
It's insanely wasteful, right?
You're burning ATP to do this.
So, the cell invests an enormous
amount in protein synthesis.
OK, which is one of the
reasons why cells hate doing
overexpression if
they can avoid it.
OK, there's a huge
selection against, you know,
when we do protein
overexpression in the lab
and turn cells into factories
for producing proteins,
they would love to be
able to avoid doing
that effort if they could.
OK. It is a huge amount
of wasted effort here.
ATP is getting burned.
OK. Great question.
Other questions?
OK. So I told you that in
bacteria, in prokaryotes,
they all end with an
N-formyl group appended
to the N-terminus.
There's an enzyme called
peptide D-formylase
that hydrolyzes off
this N-formyl group.
And in eukaryotes, humans,
oftentimes the start methionine
is hydrolyzed off using
methionine amino peptidase.
This is simply a protease that
hydrolyzes the amide bond here.
OK. So it gets in there,
hydrolyzes that amide bond
but does it specifically
on the N-termini proteins.
Turns out this is also
a potential target
for antibiotics.
So for example on the antibio--
the natural product
fumagillin inhibits angiogenesis
which is the growth of blood
vessels in human bodies.
And it does this by using a
very interesting mechanism.
So, the natural product
naturally has a three-membered
ring, an epoxide that is
precisely positioned next
to a nucleophilic
imidazole functionality.
Recall the imidazole
functionality,
we talked about it on Tuesday
in the context of RNAs.
Here, we're seeing it
again in an enzyme--
a different enzyme active site.
It's also neutral.
It also has-- this has again,
the pKa of 7 that we saw.
And so, therefore, a lone pair
is likely available to act
as a nucleophile and
be covalently modified
when it attacks this epoxide.
OK. So this is an example
of a suicide inhibitor.
It's suicidal because
it gets in and then it--
well, in this case
it's sort of reversible
but oftentimes irreversibly
modifies the enzyme active site,
and in doing so kills
the enzyme.
OK. I actually personally hate
that word suicide inhibitor.
I actually prefer
Trojan horse inhibitor
which is a better word.
It was coined by Conrad
Block [assumed spelling],
who is a little bit
one of my heroes.
So anyway I'm a-- but it's
caught on, so it's hard to do.
OK. Now, why would you want
to inhibit angiogenesis?
Blood vessel growth is great.
If you're at the gym working
out, you certainly want
to have blood vessel
growth to feed those muscles
that you're building, right?
OK. Now, the problem is
when a tumor start to grow,
they have a voracious appetite.
They are desperate
for everything.
They need more nutrients.
They need more oxygen.
They-- they're really
hungry, OK.
And so, they will attract
blood vessel growth to them
to feed the resulting tumor.
So an important anticancer
strategy targets
that blood vessel growth
and prevents the blood
vessels from growing.
And those drugs are called
anti-angiogenesis drugs.
They inhibit angiogenesis.
And for some reason inhibiting
methionine aminopeptidase is a
strategy for blocking
angiogenesis
to block the feeding of
tumors preventing a growth
and hopefully getting
them to shrivel up.
And it turns out that's
actually an effective strategy
when it's combined with other
anticancer therapeutics.
OK, so in addition to
what I've shown you,
there are higher levels
of regulation taking
place inside the cell
that are regulating translation.
And these are things--
here's my favorite.
I'm just going to describe
one of the many possibilities.
My favorite is a messenger RNA
that at one end has its
ribosomal binding site,
its RBS hidden in a hairpin.
When the temperature in
the cell has increased,
the Watson-Crick base pairing
of this hairpin breaks apart
exposing the ribosome binding
site and then allowing the
message to be translated.
That's really elegant.
OK, that's the kind of elegant
design that I really love.
And in theory, everyone
in the class could design
temperature sensitive sequences
that would get turned on at
specific temperatures knowing
for example the [inaudible]
rule.
OK. Now, we've talked
about how to--
how this happens in the cell.
We, chemists, are a creative
lot and we're constantly looking
for new ways to tinker
with stuff and try
to get better control over
things inside the cell.
And one really exciting
area that has been--
that has been really taken
off in the last two years
but has been applied for roughly
20 years or so is the idea
of incorporating natural
amino acids into proteins.
And so to do this, what chemical
biologists have been doing is
hijacking the naturally
occurring amino acids
synthetases that are found
in different organisms
and then co-opting them into
loading specific amino acids,
unnatural amino acids.
Oftentimes, these aminoacyl
synthetases are modified,
they're muted proteins.
So they're modified to
accept unnatural amino acids.
This is an analog of
an amino acid tyrosine
that would usually
have a hydroxyl
over here but now has an amine.
And that's really
a cool experiment
because now you can test, what
happens when I put a better base
in place of the hydroxyl?
If I put an aniline
functionality in place
of a phenol functionality.
It turns out this
is really powerful.
It's something my own
laboratory applies.
We apply it just as a tool.
There's other laboratories
that are trying
to extend it to other areas.
It's really-- it's something
I encourage you to use
in your proposals, OK.
It's basically bread and
butter technique used
in chemical biology laboratories
that eventually will spread
to biochemistry labs as well.
The thing is you can
do all kinds of stuff,
if you can incorporate
unnatural amino acids.
For example, you can incorporate
metal chelating amino acids.
Amino acids that form
covalent bonds in the presence
of UV light to form cross links.
This is an example of
a photo affinity tag,
amino acids that will react
specifically with carbohydrates
and amino acids that will form
cross links in the presence
of other functionalities
such as an azide.
So this is enormously powerful.
And again, I encourage
you to just use it.
It's a very routine
technique at this point.
OK. This is something that
actually works well enough.
I have an undergraduate
in my laboratory,
former Chem 128 student
who's doing it as we speak.
OK. And it actually
works pretty well,
and that really impresses me.
OK. Or, you know, he's basically
taking a technique that--
that is described
in the literature,
that our laboratory has
never applied before
and getting it to work.
OK. Any questions so far?
All right, I want to
switch gears again.
We've talked about translation.
We've talked about incorporation
of unnatural amino acids.
I next want to end with
the discussion of aptamers
and RNA sequences that
bind or catalyze reactions.
OK. So, it turns out
that you can make very,
very large libraries of RNA.
I mean I'm talking enormous.
You can make on the order
of 10 to the 13 to 10
to the 14th different sequences.
OK. So that's a one
followed by 14 zeros.
OK. And you can have all
those different sequences
in a little tiny Eppendorf
tube, small test tube.
And from there, you can do all
kinds of experiments on them.
OK, so for example, you
can identify RNA sequences
that might catalyze this
reaction pretty readily.
OK. And the way you would
do this is you'd set up--
so in this case, you're
looking for something
that will catalyze glycosylation
of this amine over here.
And so the way you will do
this is you'll have some
sequence appended.
And then, you look
for all of the ones
that have a sulfur incorporated
using mercury as a trap.
OK? OK. So that's
kind of the overview.
Let's look in at the details.
So, the key concept
here is that sulfur
and mercury form a very
strong bond and you could pull
out specific sequences
that happen
to have sulfur in the sequence.
OK. So here's the way
this actually works.
What you do is you start with
some random DNA sequences
where N is any of the
four DNA base pairs.
OK. So you start with the
four DNA base pairs ACG
or T in this [inaudible],
ACG or T in this
[inaudible], ACG or T here.
And you're probably wondering,
how do you possibly
synthesize 10
to the 14 different sequences.
Well, it turns out
it's very easy.
At every step in the
process you inject
in all four DNA bases during
the synthesis of the DNAs.
OK. So rather than just
adding A's, you had a mixture
of 25 percent A's, 25 percent
C's, 25 percent G's, et cetera.
So, that gives you a
random DNA sequence
on the order of 10 to the 14.
OK, so you have like 10 to
the 4 different DNA sequences.
You then use RNA polymerase.
We happen to favor one
that's used by a virus.
Viruses are very good
at getting their stuff
to the head of the line.
They're very aggressive
enzymes which makes sense.
They evolve.
They get to be really
aggressive like that.
And so, you can use
this T7 RNA polymerase
that will then convert
the DNA sequences
into random RNA sequences.
And then, you can
look for RNA sequences
that incorporate sulfur, OK?
And so here is your compound
that you're looking
for a reaction with.
If it incorporates sulfur
by some transition state,
then you can isolate
that sulfur using a bond
between mercury and sulfur.
OK. And the mercury is attached
up to some solid support
like the carbohydrate that we
saw earlier, like the cellulose
that we saw earlier
when we talked
about the polyT column,
exact same idea.
OK. So now the only RNAs that
will get isolated are the ones
that have sulfur incorporated
that have react specifically
with this compound.
So you go from 10 to the 14th
just down to I don't know,
20 or 30 that are
doing something.
This is really powerful
because if you get 10 to--
if you get a trillion
or a hundred trillion
different sequences together,
there's a good chance that
you can find one or two
that do something
special in your sequence.
And you can imagine
evolving this.
You could take that sequence,
mutate it further, make changes
down here, redo the selection
and then do it a bunch of times.
In practice, we often go
through like ten rounds
with these RNA libraries.
And these are often
called aptamers.
They are RNA sequences
that bind to some target.
The inventor of this whole
idea is going to be here
at UC Irvine next week.
OK, so some of the pioneers
in this area are famous
here at UC Irvine.
And a guy who is the president
and CEO of a company that set
up around this concept
would be here
at UC Irvine giving
a seminar next week.
I'll send you the details.
I encourage you to
go to a seminar.
It's going to be big.
He is kind of a heavy
weight in the field.
OK. Last thought.
There's an antibiotic called
puromycin which manages to sneak
in to the ribosome and
form covalent bonds
by mimicking the aminoacyl tRNA.
OK. I don't know why
I have this here.
It doesn't look so
much [inaudible].
Let's skip that.
OK. Let's just end
here on aptamers.
So when we come back next
time we'll be talking
about my favorite
topic, proteins.
------------------------------7ae4f1994868--
