Marty has an extraordinary pedigree.

Received here for yes the University
of Michigan for Ph D. in Berkeley

spent some time in Roger Kornberg
plant workers were right

before you become one of their work and

Dr Baxter on the hammerhead
ribozyme her research interest

from one thousand nine hundred seven
she was at the Medical Center and

she was an early pioneer in our R.
and he had some we were became

really have the vision to pick the right
people for my allergy and since I'm going

to the Scripps Research Institute
there again for research or something

else to hold a significant
number of branches on

R.N.A. she was on
the editorial board of J.T.

she was an associate editor
is an associate editor or

things she should share of and
I study section thankless task.

I do you know afraid but that is also
just a giveaway you know record.

She's shared number of meetings
organized the fight against abortion

come coupled with has
the appropriate pedigree

has impeccable publication things
you want to see in the world.

So it's a real pleasure
to welcome already.

We're going to be telling us about
our earnings I'm just pulling for

function itself more.

Thank you very much.

It's a real pleasure to be here.

I've had a wonderful time interacting with
some of you last night of this morning and

look forward to meeting more of you
today and talking about science.

So I'm going to tell you about today.

It's really I've got to the stage in
my career I have to kind of pick and

choose what I want to talk about and I'm.

Telling you about some new things today so
if I stop making sense.

Please feel free to interrupt me and
ask me for a better explanation but

essentially I'm going to focus on
two areas that we work on in my lab

one is trying to understand how our
in a can function as a catalyst so

we know that most modern day
enzymes are made of protein and

it's still a rather large mystery how
our IN A is able on a chemical level to

accelerate biological transformation so
I'll be talking about that in the course

of setting up our experiments to look
at our naked talents as we realize that

we first had to make a ribozyme that new
was supposed to fold into a ribozyme and

we inadvertently had to become experts
in our name folding and we found a way

to use that information about ribozyme for
having to probe mechanisms of R.N.A.

assembly in vivo So that's what I'll talk
about in the second part of my talk today.

So this is meant to introduce you to my
favorite R.N.A. and I thought I would

have knocked this off maybe four years
into my system professorship but

I'm still working on the hairpin ribozyme
which is a self cleaving R.N.A..

That catalyzes a reversible Faso diaster
cleavage reaction that illustrated here so

the ribozyme itself consists of
two helix loop helix elements that

are joined in nature by a four
way he'll call junction in

a minimal form of the hairpin
ribozyme contains just this A and B.

helix loopy elements joined in the in
a two way healers will John action.

This is the reaction that it
catalyzes which Cleaves a fox.

What if their bond here through
a mechanism that involves nuclear

filk attack of a two prime hydroxyl on
phosphorus to generate this train going to

go by pyramidal transition
state that is then resolved

by breaking the five prime oxygen
phosphorus Bond into products that have

to prime three prime seqlock phosphate and
five prime hydroxyl Termini.

This is the same reaction catalyzed
by Robin nucleus a protein and

Sime that is carries
out degradative R.N.A.

hydrolysis I'm sorry it's not hydrolysis
transistor for cation there's no water

at it we were very fortunate in that our
colleague Adrian Ferrie Demario was one of

the first to successfully crystallize on a
enzyme in its active confirmation and here

is the first of many views of the ribozyme
active study that I'll be showing you.

And this structural framework has been
invaluable for the mechanistic and

I'm ology that I'll be
talking to you about.

So if I mention this identical reaction
same chemistry is catalyzed by a protein

and sign that it's a textbook example
of general acid base could tell us is

the mechanism arriving nuclei say
there is one with fifteen residue and

it's on protein mated form X.
is a general base to abstract this proton

from the two prime hydroxyl to activate it
for nucleus Phillip attack on phosphorus.

A second histidine in
its present mated form

provides general acid cattell system
donating its proton to the five prime

oxygen during breaking of this
five prime oxygen phosphorus bond.

And there is also the epsilon
amino group ricing residue

whose positive charge helps to stabilize
the negative charge that accumulates

in this transition state in which
five electro negative Oxygen's

are transiently associated with
phosphorus So here we have examples of

a textbook example of a concerted
general acid base could tell.

So it's an electrostatic PATEL So
it's by a positively charged F.

one amino Now when you look at how our
name might be to mediate this reaction

it's really kind of baffling because
our ne doesn't have any functional

groups that are either willing or
able willing and

able to mediate proton transfer
at neutral ph because the P.K.

is of nucleotide functional
group ride ph you.

Stream so when we run out of thing for

example the want want to see it has
a proton at the end one position.

It doesn't want to give it up until

you reach a PH above nine point two
doesn't want to accept a proton at the end

seven position until you get to
a PH a low point two point one.

So a neutral Ph.

There are no functional groups
that could readily substitute for

histidine in mediating general
acid base could tell us.

Likewise there are no positively
charged functional groups and

rubber nucleotides a neutral ph that
might substitute for a life scene.

So the idea that really held great

sway were in around one
thousand nine hundred three.

I would say there are a whole slew of
review articles published thing that

rivals items of a new Metallica and signs
and this was based on the Cheshire Cat

hypothesis named by Michael Harris
who had had taken note

of the observation that all Arnie enzymes
are an activated by metal cat I He later.

Hypothesized that Arny's

might simply provide scaffolds for
catalytic metals so metals could act as

Louis acids to activate nuclear files or
stabilize the negative charge on lead.

Metal bound water might mediate
proton transfer So once this idea

Rose we all were pretty smugly satisfied
that we figured out that R.N.A.

could tell us this problem and then I
was able to throw a monkey wrench into

the works with the observation
that COBOL Hex I mean supports

hairpin ribozyme Fatalis actually
better then met magnesium cat ions and

this was rather startling because
I mean really guns of cobalt and

cobalt Hex I mean are inert they're
not able to exchange with functional

groups of phosphates or ribosome or

are form complexes with water
to mediate proton transfer so.

That's the only explanation for
the ability of COBOL techs I mean to

support helping ribozyme cattell So
this is like direct metal cat and

chlorination is not part of the catalytic
mechanism for this right this time.

And to date.

There are well bits of it.

So the idea is that these
cabins are required and the and

the reaction isn't activated by metal cat
eye and Keely Toure's because counter ions

are needed to stabilize the fossil
diaster backbone negative charge and

allow functional structure to
assemble in a compact form and

so removing metal can ions
disrupt the structure but

doesn't participate directly
in catalytic chemistry.

So this leads us to explain how R.N.A.
does catalyze this reaction.

And the solution to the crystal
structure shown here and

consistent with the biochemical
observations there is no metal located in

the active site and this is something that
the helping ribozyme has in common with

the pep to the transferees
center of the ribosome So

although self splicing and T.R.A.

processing enzymes probably do use
metal cat eye independent mechanisms

self cleaving our Nase and pet poodle
transfer are catalyzed by R.N.A.

in the absence of metal cat ions but here
we see that the active site is composed of

nucleotide functional
groups that in fact a raid.

In a position similar to the functional
groups the right of a nucleus a.

And there are a number of
conserve nucleotides that whose

elimination it activates to tell us
when we set out to understand the role

of these conservatively tied using a Basic
when I was in activation strategy.

In this series of experiments we
replace an active side nucleotide

base with a hydrogen atom substituting
in an age basic residue that

maintains the continuity of the day after
a backbone just leaves a solvent filled

cavity in place of the base and
when we look around the active site.

We have the nerve that there
are different levels of an activation for

a basic substitution of different
residues so ga loss of GA reduces

catalytic activity cleavage activity eight
hundred fifty four loss of a thirty.

Reduces catalytic activity fourteen
thousand fold the loss of a nine

back here reduces activity only four fold
so this is actually great news that we

saw a range of inactivating effects
because that suggests that pulling out

a single nucleotide doesn't simply cause
the entire active site to collapse and

we know what day it is when we find it if
we eliminate Ron this nucleotide here.

We can't we can't detect any
residual catalytic activity at all.

You can see it here and
this is a something that we probed more

in more detail but
I won't be discussing further today but G.

plus Ron is the nucleotide that is
the lynchpin of the tertiary structure G.

plus one forms an interaction
with the cytosine.

But polls.

The whole.

Fossil diaster backbone out
of an a form helix geometry

into this in a line configuration
more similar to the transition state.

So a lot of G. plus one is totally in
activating pointing to the important role

of positioning in orientation and
could tell sis and

I'm going to leave it at that but
no I just want to tell you that we think

these other modifications are left
ability and they probably maintain some

of the important positioning in
orientation elements of the active site.

So.

Since we have probably maintained
an active site structure

with these Dave basics of detentions
we had the idea that we could probably.

The role of these specifically ties and

Khattab also through an exogamous nuclear
base risky strategy in this strategy

we felt that we could provide
nuclear bases in solution and

that might fit into the cavity
left by the a basic substitution.

And by surveying a large
number of small molecules.

For their ability to rescue activity
by binding in this pocket left

by the missing base.

We could find out what they have
in common and maybe learn what's

important about being grown a scene or
adding in the act of sight so

this is our friend actually the first
sampling of small molecules that we tried

we basically borrowed everything we could
from our neighbors that had carbons and

nitrogen and could be dissolved in
water and of the survey we found for

the or nucleotide base analogues
that could rescue activity

shown here and in some cases
rescue in fact a similar set of

base analogues rescued both the G.
eight and an eight

thirty eight basic time and in some
cases rescue can be quite effective.

So in this case.

Iso cytosine added in solution is
able to restore activity within

about three to five fold of the activity
of the while type ribozyme So

this argues we're oppressed that
ribozymes can catalyze reactions at all.

The fact that we can get to a similar rate
within exogamous nuclear base suggest that

the exotic is nuclear base really is
restoring the normal mechanism of

the tallis it's not creating an entirely
new way of catalyzing this reaction.

A lot of these rescuing bases have
in common they all share this

Amity group similar to the robots and
could cut it in by.

Being face of the guanine and
admitting faces that they're rescuing so

that exile sickly can mean an alpha
position to a ring nitrogen

exoticness nuclear base
with Quito group but

no eggs that could mean doesn't risk
you for a permit to know so this

also lends credence to the idea that these
exotic safely base their fitting back into

this pocket in a similar orientation
to the basis that they're replacing.

So not only so purine

period with no exit cyclical
mean that competes for

rescue by any of the by side is
the nicest cytosine to sixteen appearing

in to mean appearing but
it's not able to restore activity.

And in fact if we look at competitive
in the Bishan of a rescue reaction by

appearing we can calculate an A K I that's
almost identical to the K D that we

measure from the concentration dependent
so cytosine are sighted thing rescue.

This tells us that Putin is able
to make use of all the energy from

ground state binding interactions
of the exotic it's nuclear base and

that binding alone doesn't restore
activity so it's not a matter of plugging

a plane or group into this cavity and
we're storing active site structure we

actually need interactions with this
Amity ingroup in the active site.

So there's still a begs the question of
what is the chemistry mediate mediated

by this interaction with the am
eating group in the active site.

You know as I mentioned Robyn nucleus
a the protein enzyme that catalyzes

the same reaction as it was a textbook
example of general at the base to tell us.

So the model that arose immediately from
the structure is the G eight and eight.

Thirty eight substitute for
histidine residues and carry out general.

Asset base could tell from this looks
beautiful from a structural point of view

here we see and won of G eight.

Within hydrogen bonding distance of
the two prime hydroxyl that's going to

be the nucleus.

We see and

want of a thirty eight within hydrogen
bonding distance of the five prime oxygen.

That's going to become the leaving crew.

But this makes horrible sense
from a biochemist point of view.

So this was my dream scenario
because Ronnie has a P.K.

for Deep Throat mation of that and
one nitrogen

of nine point two So a neutral PH It
already has a proton and it doesn't want

to give it up to accept another
proton from the two prime hydroxyl.

And if it did give up this proton
it would then be an ionic and

this would not this would make this
hydrogen bonding interaction with

the electro negative transition
state not favorable for

cattell says so in fact my neighbor
Albert Moser came over and

told me that he had heard that when I was
I'm ologist we're talking about GA doing

general basically tell for the hope that
I remembered my freshman chemistry and

that this was really totally implausible
from a chemical point of view.

And I had to swear that I wasn't one of
the people who had made up this model but

there is the possibility still that
there's something about the context of

the act of sight that has altered the P.K.
for guanine and

as they should if the in one position for
example closely bound metal cat

on my stabilize negative charge and make
it more easy to remove the proton from and

one but nonetheless this is a hypothesis
that needed testing in the way that

mechanistic in Somalia is generally look
at general acid basically tells us is by

measuring a PH weight profile and this
is the Ph the pendants of the cleavage

reaction catalyzed by the hair pin
ribozyme and we see a functional P.K.

of six point five which doesn't
match up with any of the P.K..

Nucleotide functional
groups that we know about.

So it's possible that there is
something about the context but

it's shifted the P.K. and in fact this
increasing rate is reflecting an increase

in the concentration of the D. pronated
probably want to mean when we look

at the PH rate profile for arrives on
time within a basic substitution of G.

eight we see a loss of activity on the
order of a thousand fold across the entire

ph range but it's the residual
activity that's left although it's

much lower it shows an identical ph
dependence on whatever is responsible for

that rate limiting step
up as a function of Ph.

It can't be GA because we see the same
ph dependence when the ribozyme doesn't

have G eight when we do
a corresponding series of experiments.

So what could be doing it still
could be media I have to say.

Technically it could be mediating
general basic TALF with but that that

step is not rate determining it's not
making a large catalytic contribution.

Well look at simply be providing a lecture
static stabilization through hydrogen

bonding interactions with the transmission
state and also helping to position and

orient on bridging and to Prime hydroxyl
oxygen from the transition state.

OK so now when we do the same experiment
with a thirty eight which was

postulated to be a general acid
catalyst in this reaction we see

that again we lose all activity or
we lose a lot of activity

across the entire ph range but
the activity that's left does have

a dramatically shifted ph dependence
we go from a functional P.

of six point five in the well type I
was I'm to one of nine point two So

whatever's going on and I have some
thoughts about what that's responsible for

this new ph dependence
that I won't go into the.

Whatever's going on in the PH pen
a step down is involved a thirty eight.

So that's consistent with the idea that
eight thirty eight is meeting General acid

to tell us this but it's also
consistent with some other ideas and

this is the principle of
kinetic ambiguity and

that is that different mechanisms
can have identical ph dependencies.

So we could see the same dependance on ph.

If the rate is proportional to
the fraction of an approach needed adenine

which would not be doing
general acid could tell.

Or if the rate is proportional to
the fraction of proto native adenine

process hydroxide ion and
I won't go through the algebra too much.

This is a familiar concept
to protein mechanised again

demolishes which the ribozyme
knowledge is all when in

fact we haven't all processes
information carefully.

But when you see a PH rate
profile you don't know

where the protests are you have
to think about the whole system.

So the mechanism that in
the mechanism in which the rate

depends on the UN pronated form of
a thirty we would attribute a rule

an electrostatic stabilisation to these
hydrogen bonding interactions and

a mechanism in which the rate
is proportional to the fraction

of proto needed add meaning and hydroxide
time we would say proto native adenine is

mediating general acid callouses by
donating a proton to the five prime

hydroxyl while in the meantime
hydroxide is doing this.

Specific based stuff in this case and we
can't tell the difference from PH weight

profiles alone which we will a thirty
eight has said no I want to tell you for

the first time about some of
the work we've been done.

We've been doing to try and

look directly at the ionization state
of these active site nucleotide so

that we can begin to distinguish these
alternative mechanisms in this room we've

been doing in collaboration with Lincoln
Scott who has made us some nucleotide.

That are these nucleotide
analogues are identical

to their Watson Crick counterparts
in the Watson Crick face but

they have a single nitrogen substitution
for say carbon at the eight position and

they have this wonderful property
that they're strongly fluorescent

in a way that depends on
the proto nation state of and won.

So it is it wanting to
strongly fluorescent when and

one is in a deep pronated state but weakly
fluorescent one N one is pronated and

likewise aged eight
adenosine it is strongly for

us and when adding is in it's natural and
pronated stated and

one that only weakly for us and when it's
made it are similar to other nucleotide

fluorescent nucleotide analogues
that have been used in fluorescent.

The fluorescent emission
intensity depends on context and

we had to be very careful to make sure we
could still see some fluorescents when

we put the analog in the context
of a structured Darnay And

here you see an experiment where
you take in eight days a G.

in a single stranded R.N.A. and
titrated in either a perfect complement.

Or a strand that is
partially complimentary.

And in fact this teal looks
represents the domain

A of the hair can rub his time and
we see that we do

commission intensity as we
increase complex formation.

And this reduction in the mission
intensity is a little bit stronger for

the perfect duplex than for the bold
duplex consistent with the behavior that

people have seen for similar structured
Arnie's we don't have enough.

Mission intensity left in
the structure to look at.

So we were able to use the PH
dependent supplier essence

to to measure the P.K.
for eight days of wanting the car.

Text of single stranded double
stranded in Boulder duplex R.N.A.

And as you can imagine when a days a G.
has a job in forming

a base pair with a cytosine it's much more
difficult to remove its pro time the P.K.

shift into the basic region and the G.

in the context of
the hairpin domain domain

A of the hairpin arrives I'm a somewhat
intermediate between a perfect duplex and

the bald duplex so it's a little bit
easier to part of the proton offline and

domain name so the big question is what
is the P.K. for the Swanee in the active

side of the airplane where I was I mean is
that shifted into the neutral range where

it would be well suited to do
general at the base could tell.

And the answer is no.

So this is the last week
we see the Domain A alone

functional hairpin ribozyme with an active
display virtually identical P.K.

for nation and want to think
on their far in the basically.

Interestingly when we begin titrating
magnesium into this reaction in the open

symbols here we see that the P.K. begins
to shift more into the acidic region as

you would expect if there's an increasing
positive charge in the vicinity.

That's able to neutralize the negative
charge that results from piling a proton

off the in one position of adenine So we
do see metal dependent shift in the P.K.

toward the neutral range but
nowhere near the totally neutral P.K.

there would be consistent with for
GA in general based

identical experiments for
ADD meaner underway and

we now have to add mean incorporated into
these molecules we haven't yet put into

a full length intact ribozyme but they are
the prediction is very different depending

on whether ad name is functioning
as a general athlete good talent.

Or whether it's providing
a lot of static stabilization.

OK so what just to summarize
this part of my talk so

far we know that catalytic chemistry
doesn't always require metal cofactor So

our job now is to figure out
exactly how functional groups that

are in a contributing contribute to
tell us as we learn that the Amity of G.

eight May thirty eight interact
in the transition stage.

We're still sorting out whether that
mechanism is primarily electrostatic

stabilisation or proton transfer and of
course all these functional groups have to

have rules and positioning in
orienting this active site and

then finally what we've just
learned is there is no real proof

of the ionization equilibrium of
G eight in the context of that.

OK now I'm going to shift gears a lot and
tell you

the use we've made of all the problems we
encounter trying to assemble functional

ribozymes in order to begin this
mechanistic and I'm ology and I probably

don't need to really explain this but the
point of this slide is to say that R.N.A.

is have structures and in fact a single
R.N.A. can have more than one structure

over the course of its biological function
and many of the popular small are they.

These days small nuclear are nay
involved in R.N.A. interference actually

form complex or through complementary
based pairing and specific structures and

then there is the recently discovered
ally both which are in a which functions.

Many of which function through
a conformational chain exchange

reaction that accompanies finding a small
and so it's really important to understand

how our names form functional structures
and this is not an easy problem.

In fact my my post-doctoral
mentor called this alternate

compromise hell which is what traps us all
when we design an R.N.A. sequence with

the idea that it will assemble into the
structure we're interested in looking at.

And it has something else in mind.

So our name is comprised
of just four simple Bill.

Blocks and those for building blocks can
associate in sixteen sets of pairs and

eight of those pairs are stable.

So if you anyone who's run
an employment program known

that if you feed in a large R.N.A.
you get back a multitude of

structures only one of which is the one
that has a biological function.

And to make matters worse not only do
our names assemble into a multitude of

alternative structures with similar
thermodynamics stabilities but

once they formed one of these
structures they can't get back out

if you have a base pair of helix
with six or seven base pairs in it.

It could be hours or
days before that helix dissociates to give

those strands an opportunity
to sample another structure.

So this problem.

Jamie called Kinetic trapping.

So we set out to use the right is that
also how do cells get around this problem.

Maybe because we can't afford to have
ninety nine point nine percent of our

foreign aid getting trapped in
some nonfunctional structure.

How do Arnie's find a specific
functional structure interview though.

Or maybe proteins help by either
trapping functional structures or

celebrating exchange among
alternative structures.

Also we have the advantage of being able
to assemble Arnie's as they're made.

So it's possible that there is
a sequential component to assembly.

So that structures that can form among
five prime sequences are favored

over structures that require interactions
between sequences that are synthesize

first and sequences that are synthesize
later and also Jamie is not here so

I can mention this the conditions used for
in vitro experiments have

traditionally included extraordinarily
high concentrations of Diabaly on cat ion.

And to some extent we may have
created our own Khan from or

hell by working with debilitated R.N.A.

is the need a lot of structural support
in the form of dive Alun can I.

And that's the point that I'm going
to argue strongly in favor of.

So they have.

Then ribozyme inside Dealey
suited to sampling how principles

that have been elucidated through in vitro
reactions translate into an interest and

environment because we know a lot about
how structure affects the activity.

I mentioned the positives are cleavage
reactions are readily reversible and

in fact the ligation reaction is
favored over cleavage dramatically

when the hair pin ribozyme assembles
into a stable tertiary structure.

So if we drop the temperature increase
the concentration of magnesium or

if we add back for our way here
local junction to a minimal time

all of these conditions stabilize
the tertiary structure and

shift the equilibrium between cleavage
unlike Gratian of bound products

back toward like ation so stabilizing
the tertiary structure will reduce is

observed cleavage rates by promoting
legal obligation of bound products.

Also the product assembles the product and
route assembles with

the rest of the ribozyme through the base
pairing interactions in this H one helix

stabilizing that interaction
by adding base pairs or

changing the base composition.

Stabilizes product finding and
also reduces observed cleavage rates by

a keep promoting the accumulation
of ribozyme product complexes.

That then readily go back through to
reverse a cleavage reaction through real

geisha in a bound product and
we know a lot about these rate and

equilibrium constants under a variety
of conditions in vitro that we

had to fight our way through in order to
look specifically at the cleavage step in

the assembled complex we
decided that we should turn

lemons into lemonade by using
the system to ask how the.

Program matters measured in vitro
translate the behavior of this

ribozyme in.

And this is my sales pitch for why the
HIPAA drives time is such a good probe of

R.N.A. structure with a simple R.N.A.
with with well.

All known common R.N.A. most teeth are in
a structural motif duplexes he'll call

junctions certain fashion
lies treasure interactions.

We know that metal don't participate
in catalytic chemistry and

we don't require any specific co-factors
of the behavior of this ribozyme

in yeast reflects a fairly generic R.N.A.
structure and

used as well suited to these experiments
because of the tools that are available

from molecular biology and also because
M.R. in a lifetime is in the east.

I want to bacteria mammalian
cells are conducive to doing

quantitative kinetics and you'll see
the importance of it in a minute.

So this is our system we have there and

ribozyme inserted into the three prime on
translated region of the yeast P.G. K.M.R.

NE We expressed this AM R.N.A.
from a lack totem duce of the promoter and

this allows us to get a burst
of transcription galactose move

the cells into glucose in
transcription stop immediately.

We can survey the amount of
Cleveland unclever Merrick M.R.

ne using in our nation
protection essay and

I still show this slide because it took my
student a year to figure out this assaye

that I'm going to whip by at this time and
just explain that.

At steady state we see about
thirty percent of the ribozyme

in the formal on Qatar nay and seventy
percent in the form of five prime and

three prime cleavage products and this
is significant because it tells us that

cleavage occurs on the same a life time as
a lifetime of this P.G. K.M.R. named evo.

All our names turn over and sell some sort
of synthesize at a characteristic rate and

for P.G. K.M.R. ne that turns over and
through and dodginess M.R.

in a degradation pathways with a T
one half of about fifteen minutes.

The fact that we see some uncut R.N.A.
means that cleavage can't be

happening much faster than
with a T one half of fifteen.

We wouldn't have anything left on cut at
steady state clip it can't be much slower

than this normal turnover way because if
it were much slower we would see a greater

accumulation of unclear you've done it and
we realize that we should be able to use

in dodging this Deborah Deshaun rate
of the P.T. K.M.R. nay is a crock to

measure intracellular cleavage kinetics
precisely because I'm Eric M.R.

in a with a self-cleaning ribozyme I will
turn over to be in dodging this pathway.

But it also has a second pathway to
decay and that's true self cleavage.

So the overall rate of disappearance
the full length kind of merit M.R.

nay itself Cleaves it's going to be
faster than the rate of disappearance

of a kind Merican M.R. an A that has
a mutation activated ribozyme in it

by an amount that corresponds exactly
to the intracellular cleavage rate.

So we can measure intercellular cleavage
by comparing by allowing cells to

produce going to Martin galactose shutting
off transcription with glucose and

measuring the amount of polling on
a left after various times and we can

see that mutation activated primary can
marinade the case with the characteristic

of a rate of about two point zero to
four minutes shown here in blue but

arrival time that can take over an M.R.I.
The contains a self-cleaning ribozyme

I'm sorry shown here read
the self cleaving M.R.

nation in the loo case faster at
a later point zero seven per minute.

So this tells us that in for
cellular cleavage occurs at a rate

of point zero seven minus point
zero two point zero five.

We can also runs we know the intrinsic
degradation rate we can also calculate

the intracellular cleavage away from
the relative steady state abundance of

the cleaving M.R. Ney's and
I won't take the time to take you.

The algebra but when we calculate and this
is subject to a different set of artifact.

So we were relieved to see that this
calculation from steady state abundance

gave a similar answer as the decay
time for us and giving us

an average estimate of interest or
cleavage rate a point six per minute and

this rate is significantly slower than
the rate that we had measured for

self cleavage of this ribozyme in vitro
understander conditions which were about

two point three per minute and we wanted
to ask what inhibits cleavage in vivo

Why is it slower in vivo than in vitro in
our first idea was that it was mis polling

because we put it in the M.R. Nay but
has opportunities to interact with M.R.

in a sequence as maybe it's
getting stuck somewhere.

So we hauled out some of our sequence
variants that we had discarded

because of their propensity to mis fold
in vitro and thought maybe they won't or

they shouldn't.

Fortuitously have the same interactions
with M.R.S.A. flanking sequences and

to our surprise all about our names
that missed it in vitro that we had

stopped working with displayed identical
intracellular cleavage right so

this was the first indication that maybe
this confirmation battle with something we

had created for ourselves and
cells had a way around it.

So our next experiment was to try and
figure out.

Maybe the interest I were environment is
too favorable maybe we're accumulating

stable ribozyme product complex truth and
maybe a stable tertiary

interaction is driving the reactions
back through like geisha and so

to test this we sampled different
abilities of ribozyme product.

And if stable product binding were
driving rely geishas of bound products

we might expect that weakening
product finding Linux cell array of

served intercellular cleavage rate and
we didn't see that and

we saw that ribozymes a short
helix is along here with

these cleave to Berkeley the same rate in
the bow and if we change it back too far.

Lost the ability to form
a functional ribozyme at all.

So that's not the case.

Instead we decided that we had better
look at the activity of this when I was

I wonder around a conditions that are more
similar to those that existed in vitro.

And when we looked at ribozyme activity
and reaction in vitro reactions that

contained only two million molar magnesium
we saw the thing doesn't minimal

ribozyme doesn't function in physiological
ionic conditions so how does this thing.

In fact it functions even worse than
intracellular ionic conditions in

vitro than it does in the vote.

So how does this thing work in nature
where after all it has a job to do and

replication.

Well this is when we notice that
in nature the hairpin ribozyme

assembles in the context of
a fab way he'll go junction.

Maybe this far way he'll go Junction
is a compensates in some way for

unfavorable ionic conditions and sure
enough when we look at the activity of

a far away junction ribozyme
it maintains full activity

in vitro in reactions that include
physiological and a condition.

And Cleaves much better than a minimal
ribozyme in vivo So this tells

us that in fact nature is found a way to
generate R.N.A. as with significantly.

They were sufficiently stabilized
tertiary structures that they don't need

ten million dollar magnesium and
this is a theme that we've seen over and

over again after Mers created in vitro.

Under conditions with high
concentrations of diet and

one cat I don't work nearly as
well as ride both which is that

involve evolved in the EVO to bind
small molecules and rival switches

all have much larger structures
buttressed by many tertiary interactions.

So that sufficient interest I
would cleavage that we see with

a junction where I was I'm the only if
we have a product found in a small.

Number of base pairs in
this case if we do increase

product binding affinity in the context
of a four way junction ribozyme we lose

activity just as you would expect a stable
product binding promotes accumulation

of varieties on product complex and
real geisha So we've now got evidence for

this junction when i was time
functions is a very efficient and

by comparing the number of variants
one with a small number of base

pairs holding on the product to mourn with
twenty base pairs clearly on the product.

We were able to deduce kinetic mechanism
for this intercellular reaction by

assuming that one product so Station is
really fast the cleavage rate that we see

corresponds to the cleavages rate constant
where product Association is really slow.

I won't take you through the algebra again
but this gives us the way of calculating

an interest cellular geisha rate constant
from the equilibrium between cleavage

unlike ation and when we have arrived time
it displays an intermediate intracellular

cleavages we can by assuming that it
partitions between product Association and

geisha and we can calculate a rate for
a product to

the point of this I think my mother may
be the only person who really cares about

the details of these numbers point is
that the values that we measure for

cleavage unlike Asian rate constants and
product Association kinetics in vivo

correspond within about two fold with
the same parameters that we've measured in

vitro provided that are in vitro reactions
are carried out in physiological fall.

So this kinetic mechanism gives us no
indication that there's anything special

about an inch or so environment that
functions essentially as a swamp where

the programmer is of this intracellular
cleavage reaction are determined by

pictures that are intrinsic
to the structure of this.

And I think.

I'm just going to rip through this next
section to say that we've been able to

measure K. Cat and K. M.
values for Inter Milan other reactions and

and found a similar phenomenon that
finding rate constants that we measure for

substrate association with the ribozymes
are what we would observe in

vitro provided that we adjust to be
ironic conditions of corporately and

now I want to tell you this is our
interest cellular EDI Hoff's which I think

my mother was quite proud of and
probably no one else has seen before and

I want to tell you all from what I thought
I said so I it's a critical determinants

of our in a feeling pathways in nature our
names have adapted to intracellular and

conditions by generating additional
tertiary interactions to

buttress the structure and there
are examples of this after Morris versus

right both switches and virtually all
minimal ribozymes that function well and

one cat on high concentrations
of diving and can have natural

analogues that function in
physiological and a condition.

And I want to just take a few minutes to
tell you our latest stupid molecule tricks

we decided that we could use this ribozyme
to probe the mechanism of secondary

structure assembly by
inserting does designing in

an alternative structures we design them
in by simply in third inning a sequence

upstream of the ribozyme
complimentary to the ribozyme so

that it can form a stem loop
with ribozymes sequences and

that stem loop all in all H.
won is incompatible with

assembly of the essential
a twenty helix with a ribozyme So

we put a complementary insert upstream and
we put a similar insert downstream or

it can form a stem loop with
the opposite strand of the essential H.

one stem with the ribozyme and
the formation of either.

I'll take one down.

Loop is inactivating to the ribozyme

because the events assembly of
an essential ribozyme structure.

If a assemble sequentially.

That is at the five prime to three prime
nature of our nature inscription governs

the secondary structure assembly pathway
we would expect the upstream stem loop.

To be far more inhibitory than the
downstream stem loop which doesn't have

an opportunity to because the upstream
stem loop can form before the ribozyme

has been transcribed before the opposite
strand of H one is available for

kneeling or the downstream in third.

Can't form until the entire Wyvis time
has had an opportunity to assemble

a functional structure.

And if the folding outcome is determined
by thermodynamics that is the P.R.

in a finds the most stable structure but
I wrap it exchange among alternatives we

would expect both to be equally
inhibitory because we design them to be

more stable than the H. one stem of the
ribozyme that they're competing with so

when we transcribe these constructs in
vitro using T seven R.N.A. Plimer ace.

We see exactly what we would
expect from a sequential folding

model that is the five prime minster

almost completely inactivates assembly of
a functional lives that we see very little

activity this is the activity of the while
typewriters time for comparison.

Whereas the three prime inserts
displays a perfectly by phase ic

reaction kinetics where half of
the ribozyme seems to assemble

into a functional rive the time that
cleaves with the expected kinetic

half of the ribozyme gets
trapped in this three prime

pay twenty trucks were able to verify
that these structures not responsible for

the loss of activity using
chemical protection that thing.

So what's going to happen so this pattern
fits perfectly with what you would expect

if secondaries of structures
assemble a prime to three prime.

So what happens when you
put these things in vivo.

Fine both stem loops upstream and
downstream totally eliminate

any detectable activity in Bebo So
this would be on the face

of it consistent with the rapid exchange
model saying that these ribozyme live

in the EVO an environment that has rapid
exchange among alternative confirmations

and that's why there is no
evidence that sequential assembly.

But if you think back to the kinetic
mechanism that I showed you.

We found no evidence for a rapid product
Association in vivo So why don't we really

kind of just stir defeat this evidence
that the thermodynamic model seem

to explain or observations so we came
up with the thermodynamic model but

there are a chaperones that accelerate
exchange among these alternative

confirmations of till they find the most
thermodynamically favorite form.

So we came up with another model that
we call the delayed filing model and

that model postulates them in
fact our name doesn't happen

sequentially during transcription.

But because proteins are deposited
on Mesa transcripts by the cut box

the terminal tail of R.N.A. Plimer ace and

maybe pulling domains
are only become free to fold.

Only when these proteins are dissociative
and if that were the case following might

be determined kinetically it might be
that the structures that form first and

are slow took to dissociate in kinetic
terms are the ones that persist.

So there we would expect to see a kinetic
component to the folding outcome.

Too and it may be that we in our initial
construct by putting our competing

sequences making them able to
form by can take you a sequences.

They were favored kinetically
over sequences that had to form

between the distant parts
of the ribozyme sequence.

So we set up a circular.

Are you in which we opened up the other
helix at the base of the domain and

put our complimentary sequence in
there where it had to reach across

the ribozyme to interfere with assembly of
the Reivers time structure and when we set

it in we looked at the circular permits we
saw the ribozyme get to win a little bit

in the circumstance here the eighty
two want to be like is assembling for

contiguous sequences and
one helix has to fight its way in.

So this is the thermodynamic calculations
are a little complicated for this

kind of construct so we can't be totally
convinced that kinetics are winning.

But this is the outcome that
we would expect if con to

effect a somebody kinetics and.

To test whether unfailing kinetics
contribute to filling out.

We stabilized Juan and all H.

one kill season series
to ask if we made it so

stable that it would be
expected to dissociate slowly.

Could we achieve a kinetic traffic
that would not that would not

then be resolved in to but the most
thermodynamically favored outcome.

And I obviously would be telling you about
this if it hadn't worked to some of.