Ribonuclease P: A Small Step in the RNA World with Sidney Altman

(whooshing) – [Announcer] This
program is a presentation of UCTV for educational
and noncommercial use only. (bright music) – Welcome to the second of the Hitchcock Lecture series. I’m Fenyong Liu, a professor from School of Public Health and also the chair of the Graduate Group of Comparative Biochemistry. On behalf of the Dean of
the Graduate Division, as well as the Hitchcock
Selection Committee, it’s my great pleasure,
as well as my honor, to introduce today’s speaker, Professor Sidney Altman. Professor Altman is truly
a distinguished scientist and an outstanding teacher. More importantly, he’s
also a wonderful mentor as I personally experienced
as a postdoc fellow in his laboratory at Yale. I will not repeat the long
list of his accomplishments here as Professor Lester did yesterday. However, I would only want to edit that he has spent his entire
40-plus year career focused on a single, yet
critically important enzyme called Ribonuclease P or RNAse P. His scientific journey on RNAse P is an inspiring story. It starts with his initial discovery of the substrate, as well
as the enzymatic activity of the enzyme RNAse P when he was a postdoc fellow under the guidance of Sydney
Brenner and Francis Crick at MRC Laboratory of Molecular Biology in Cambridge, England. He then moved to Yale, subsequently cloning the RNA subunit of the enzyme, RNAse P and demonstrating the catalytic activity of the RNA subunit. His research has revolutionized the field of molecular biology specifically the function
of RNA molecules. Today he will give us
his personal reflection of the discovery of the catalytic RNA and share with us his
recent research progress in his laboratory. Please join me in
welcoming Professor Altman. (audience applauds) – Thank you very much, Fenyong for the introduction and for
your wonderful hospitality during my visit here. And once again, I wanna thank the members of the Hitchcock
Committee for selecting me for these lectures, and I hope I deliver
in some reasonable way. I want to start by
repeating something I said at the beginning of yesterday’s lecture which is that you’ve
all paid for my research over the past 30 or 40 years. Your income tax of which a few pennies on a dollar, so to speak, are devoted to my particular research through the National Institutes of Health and the National Science Foundation. And I want to thank all of you taxpayers for your donations over the years. I feel that’s a way of indicating my appreciation instead of showing a slide with many different names and
many different agencies on it. Okay, so yesterday we talked about the origin of life a little bit and what the RNA world was. And today I’m gonna
focus on ribonuclease P which Fenyong indicated which has a subunit that is catalytic. What I’m going to do is talk about both the RNA and the
protein subunits of RNAse P in various organisms, and I wanna indicate that RNAse P is an essential enzyme in all organisms. I won’t go through the data, but it is clear that without RNAse P these organisms don’t grow. After I talk a little bit about the enzyme I’m going to talk about
different substrates both in E. coli and in higher organisms. And then the last part
of my lecture will be on some recent experiments in my lab in which RNase P is an important mechanism in providing a drug therapy. The first slide you see is an old slide and it indicates the first substrate for RNase P. This is a tRNA molecule
as I showed you yesterday. It’s in two dimensions. And these are extra
nucleotides at one side of it. And RNase P cuts right
here at nucleotide one of the mature tRNA sequence. So all these nucleotides are removed. These nucleotides are
extra nucleotides too and they’re removed by different enzymes. So essentially we make
virtually a functional tRNA out of this cleavage here. One of the problems we had to confront and I think we solved it
as I’ll show you later is what is the specificity of this enzyme? In fact there are about 60 different tRNA precursors in every cell. None of them have a, none of them have a consensus sequence or similar sequences
around the cleavage site. And so how does this enzyme
recognize its substrate? It does recognize it by looking at a partial RNA helix on
this side of the substrate and a single-stranded
region on this side here. We have two extra base pairs here, but it’s not at all clear
that they form in vivo under the conditions
that we’re talking about. Now this is a chart which characterizes the catalytic activity of
the RNA subunit of RNase P. Let me just say although
you cannot see it clearly, it’s not shown here, the structure of the catalytic subunit, although I will show a picture of that as it has been recently crystallized by Mondragon at Northwestern University, the catalytic structure
is extremely similar in all organisms. However there is one
difference between that and the proteins in the enzyme. In E. coli bacteria, let’s say in E. coli and
in bacteria generally, there’s one RNA subunit which is catalytic and there’s one very
small protein subunit. If we can rate the catalytic
activity in bacteria, let’s say it’s of the
order of one to 10 here. Then we have other species
involve the Archaea which we now classify as
a separate kingdom here, and the Eukarya or eukaryotes. In Archaea there’s one RNA subunit and four and possibly
some cases more proteins in the enzyme. The RNA subunit is catalytic and in several cases tested. But the catalytic activity
is only about 1/10 to 1/100 of that compared to that in E. coli, but it’s easily recognizable. And this is at pH six where in fact there’s very
little degradation of the RNA as you incubate it in a reaction mixture so that you can run
reaction times over night at pH six under the conditions Leif Kirsebom in Sweden really devised. And you can easily see very
low amounts of activity. And Kirsebom and his group found that the RNA from a couple of Eukarya, as there’s one RNA subunit and there are nine or
more protein subunits. The catalytic activity here
is about 10 to the minus five compared to the catalytic activity here. So these are measurements that have
been made rather carefully, so we’re very confident of these numbers. This indicates the uncatalyzed reaction. So if you just let the
substrate sit around, you’ll find the cleavage rate is around 10 to the minus nine or so. And here are the relative references: Guerrier-Takada et al. in E. coli, Pannuci et al. in at
least one of the Archaea, and Kikovska and Kirsebom
et al. on eukaryotes. I think that’s all I’m
gonna say about the RNA at the moment. Now we’re gonna talk about diversity in the subunit composition of RNAse P. So we have in bacteria one protein which we call, we had labeled a long time ago C5 protein because it just of its position in gels. In Archaea there are
four different proteins, and now another one has been characterized which is actually a ribosomal subunit which when added to these four stimulates the activity considerably. And it has some homology with one of the proteins found in yeast and in HeLa cells. And we have in HeLa cells, this is a human enzyme, 10 subunits here and you can see some of them have homology
to the Archaea subunits. Here’s RPP30. Here, RPP38. Here et cetera, RPP21 and 29 and POP5 here. But there’s several subunits here and as I indicated from
the previous slide, they appear to contribute
to the catalytic activity. And by saying appear to contribute, it could very well mean stimulating the RNA activity by itself by adding these various proteins. So I should also say something about the evolution of the RNA subunit. The shape as you can draw
in at least two dimensions is very similar from
bacteria up to human cells. There are some differences,
generally it’s about, the RNA’s about 350 nucleotides in size. But there are a couple of yeasts where it’s 250 nucleotides in size and there are a couple of other organisms where it’s like 800 or
900 nucleotides in size. However the difference in size corresponds to a difference
in the recognition of certain subunits, and does not seem to affect
generally what we observe. The catalytic portion of the enzyme remains more or less constant although it does change somewhat between the Eukarya and bacteria. And, these slides are from Venkat Gopalan at Ohio State University. And this gives an
indication of the evolution of the protein subunits. We have a common ancestor here where presumably RNase P
lacked protein subunits. Although there’s no direct evidence, there’s nothing to indicate that that should not be the case, we know that if we isolate
the RNA subunit by itself in test tubes, in vitro, it has the catalytic
activity that is for bacteria and we can show it for
these other organisms. And then for bacteria there’s the origin of a single protein cofactor. And for the other organisms here apparently you start sharing
four protein subunits, and then you have the Archaea. And the Eukarya are out here. So this is a rough picture of
the evolution of this enzyme in terms, if I may make the reference to what I talked about yesterday, this is an indication of
the complexity of evolution as it goes along. So let’s not think about humans as thinking individuals for a moment, but if we just look the structure of a key enzyme, in this case, and determine what goes on, the bacterias are very simple, but eukaroytes are quite complicated in this particular case. And we see that the proteins
seem to have replaced the contribution of RNA in
terms of the catalytic activity. Now I’m gonna talk about some substrates that have been found in E. coli to date, and some of them exist in
other bacteria as well. First, we have tRNA precursors
which are listed here and as I said there are
about 60 different precursors in any particular organism,
bacteria or eukaryotes. Then we have 4.5 sRNA in which the structure
can be drawn like this of long double-stranded regions, a long hairpin. But the precursor has
extra nucleotides here and the arrow indicates
where E. coli will cleave it just at this point here. So this in fact was good reason to believe
that our other data on the structural
features that are required for cleavage by RNase P is correct. That is to say, we have a long period of double-stranded RNA here of which actually you
only need about a half a helical strand of, a helical twist of RNA, and a single-stranded region here. This is another similar
substrate from E. coli. Here are others, I won’t go into what they’re doing at the moment. This is a phage RNA actually. This is actually tmRNA or an RNA which is both a messenger RNA and looks like a tRNA. That is to say, there’s a coding region in this region here and it codes for a small protein of about 11 amino acids which tags proteins
that are to be degraded inside E. coli and other organisms. But the ends of the molecule look just like a tRNA molecule and it has a precursor sequence here, and RNA cleaves it right here. So these are all important
molecules in E. coli, and they have, they are not unstable molecules. They have reasonable
lifetimes inside cells. And if you know how to look for them, and now we know how to look for these, you can find them quite easily. Several years ago, about seven or eight years ago, in my lab it was found that there was another set of substrates for RNase P. And it remains to be shown by
really rigorous experiments that this does function
in gene regulation. But we’ve shown in
model systems that does. So here are various operons indicated. An operon is a region that has three genes usually coding for similar
metabolic function, but not always. Here’s another one here with five genes. Here’s the histidine
operon with several genes. And the fat arrows are
coding regions for proteins. And these narrow lines here are the regions between proteins. What we can say, for example, is that RNase P, in these cases, cleaves in the regions
between the coding regions. So these underlines
indicate cleavage here. One here. One here, and one in this region right here. We isolated these
molecules just by looking at a temperature-sensitive mutant in RNase P at a high temperature and we picked these molecules
out from many molecules that you could find under these conditions that did not appear under
normal growth conditions. So these are some examples
of cleavage in our operon, but it’s much easier to
do things in an operon that you really recognize which is the lac operon for the metabolism of lactose. And here we have the lac operon. There are three genes: lacZ, lacY, lacA. We know what each one of these codes for, and here are the intergenic
regions right here. So here’s the end of the lacY region in this particular case. So we’re looking at this region in here. And this is the beginning
of the lacA operon in UUG. This is a UUA here. And this is what the region between the two coding proteins looks like. And it looks like it
actually has a structure that would be recognized by RNAse P. And in fact it does. This where P cleaves, it’s right here. I won’t go into data, but this just indicates
what I’m talking about. That is to say, if we put this structure on a piece of genetic
information where it’s coded for in a bacterium where we can turn off RNase P when we want to, and we can do that, we show that when we turn this off we make more of lacA. When we allow P to function, we make much less of lacA because you get cleavage here and degradation under these cases. Now it was noticed in the ’60s when RNase P, ah excuse me, when the lacZ operon was
being studied by many people that there was a significant difference between the translation or the production of these three genes here. In fact there was much less of lacA made than lacY or lacZ. And there was really no
explanations at all given for that. I think in this case, with
the model system we have actually supplied that explanation. But this simply gives
you another indicator of the range and variety of substrates
for RNase P in bacteria. So we can talk a little bit
about now in eukaryotes. David Spector some time ago published a paper in RNA, I think it was 2000, it was either early in 2010, ah, yes it was. And he asked us for samples
of RNase P from HeLa cells which we sent to him. He was looking at a substrate that looked like this. We don’t have to worry about most of this. This is just taken from his paper. In fact, he was looking
at a very long piece of messenger RNA and inside the message he identified what looked like a tRNA molecule here. And as it turns out, RNase P does cleave this molecule right at the junction of the single and double-stranded region here and you wind up with this, okay. This describes further
events in the pathway which I’m not interested in
for our purposes at the moment. But he clearly had found that RNase P does cut in that particular position, and he recently told
me that he has at least one other such case of substrates that look like this in eukaryotes. So that’s satisfying. We already knew from previous
experiments of course that RNase P cut tRNA precursors, for example, in HeLa cells or mouse cells or any other organism that
you wanted to look at. We know that RNase P is
actually located in the nucleus of Eukarya and I might
mention that a little later. So it cleaves tRNA precursors. It cleaves these long messages with tRNAs in the middle of them. We have evidence and we published evidence that it cleaves ribosomal RNA precursors in HeLa cells and in yeast. It’s hard to get purified
ribosomal precursors, but the evidence we have
is pretty convincing. So we have at least three classes of substrates in eukaryotes and I’m sure there are more substrates. It’s just that we haven’t, and I don’t think anybody has made the effort to look for more. Now it had been suggested for some time that in various organelles in eukaryotes, for example, mitochondria, or a chloroplast for example, that there was no RNAse P of
the kind that I’ve mentioned. It had been indicated
that there was RNAse P that contained no RNA at all. However a recent paper by
Koehler and Teitell in Cell this year, a few months ago, actually was quite a remarkable paper. It was on the function of an enzyme, polynucleotide phosphorylase, PNPase which is responsible for importing various pieces of RNA into mitochondria in human cells. And one of the things
it imports is RNase P. And in fact, Teitell and
Koehler and their co-workers showed that there were certain sequences that actually were cleaved
by the conventional RNase P. Now these diagrams are
taken from another paper, but it doesn’t really matter, they demonstrate what I wanted to show. And that is when you have
the classical RNase P with an RNA subunit, and in mitochondria you have situations where you have a long piece of RNA and interior in the RNA there
might be two tRNA sequences. You have messenger RNA here, messenger RNA here. And in these kinds of
cases RNase P cuts it here at the beginning of this tRNA, and cuts it here at the
beginning of this tRNA. It had been shown by people who worked on these enzymes that supposedly don’t have
RNA associated with them that if you have an RNA like this at the head of a region where
there’s a messenger RNA here and you have one RNA here that that these other enzymes cleave this RNA at this particular point. So that is a remarkable new finding. I found that the whole
paper that I looked at in this case was a very interesting paper and very useful and it showed in fact that RNase P seems to be, in quotations, everywhere, the classical RNase P. There’s one other thing
I’d like to show you about the structure of the enzyme and it’s complicated. It’s essentially the crystal structure, a fantastic crystal structure performed by Mondragon and his colleagues at Northwestern University. They have made, they have done, they have solved the crystal structure of the ternary complex which contains the RNA
subunit of RNase P from a bacterium that’s not too
different from E. coli, the protein subunit of the complex, the tRNA product of the complex, and a single-strand piece of RNA which supposedly would represent the cleavage product
from a tRNA precursor. So effectively we’re looking
at the product of the reaction with the product in the substrate. This paper will be
published in Nature soon. I can effectively guarantee that. Okay, so this is a different
way of looking at it from what the conventional picture is, but here we have in blue or purple, whatever you want to call it, the RNase P RNA. It’s organized in this particular way. Usually we turn at least by 90 degrees and look at it from that point of view, but that’s all right. And I’m not going to to into any of the crystallographic
details at the moment. I simply want to show that this is what has been found and how it confirms some of the predictions that were earlier made. This is a tRNA precursor here. So this is the anticodon stem down here. And this is the recognition site here at the junction of the tRNA molecule. And there would be a
single-stranded region which we can’t identify here. The protein cofactor is
located in this region here. Now there has been a lot
of RNA work done on this before the crystal structure. So we already knew something about which parts of the RNA were
in contact with the protein. We knew by cross-linking
studies which part of tRNA were in contact with this
piece of the RNA too. Nevertheless, I think this is a fantastic piece of crystallography and very interesting work. So I offer my
congratulations to Mondragon. We had shown that the RNase P in
complex with the substrate has different confirmations. It can be the transient state or the active state of the enzyme and we don’t know anything about that yet. It could be the free substrate or it could be the cleaved product in the single-stranded region. But we had shown from various
enzymological experiments in vitro that these three sites, these three confirmations must exist. And Mondragon has succeeded in producing the crystal structure of this one. We had also shown from cross-linking and other studies that the aminocyl helix of
the substrate is denatured during the cleavage product. That is to say, at the helical end of the tRNA precursor where usually at the three-prime end an amino acid is attached, at the five-prime end you
have an extra substrate, those strands come apart. So it’s no longer a double-stranded
region in the helix. And we’d shown that that was the case. And if you cross-link the
helix and it could not denature then you got no enzymatic reaction at all. And that was confirmed by
this particular structure. It was shown by Mondragon that in fact the two parts of this helix are denatured in the complex. And furthermore the
position of the protein has been specified by various methods and the crystal structure
confirmed all of that too. So it’s quite satisfying. Okay. I’m going to go on now to
some recent work in our lab and I might skip some stuff because I don’t wanna go over time. First of all, here is the classical tRNA precursor. It was from these molecules that in fact we first deleted this region here, this stem and loop and this stem and loop. It was done with Bill MacLean and we covalently linked this to this and that was a very good substrate. It was just one continuous
helix with a loop. And then Tony Forster, a
postdoc from Australia, this is 15 years ago at least succeeded in deleting this loop here. So we’re just left with this strand hydrogen bonded to this strand here. And if you draw that differently, this is what you see. In fact these are now two
different RNA molecules, but they get cleaved very well by RNase P at this point here. And they’ve been drawn
in different colors here, because now they represent
the basis of the therapy I’m going to talk about. This can be any target RNA in a cell, any cell. And as long as you have
another piece of RNA that can hydrogen bond to it here, it’s especially true of
bacteria in this case, we call this external
guide sequence, the EGS. And as long as this
can hydrogen bond here, this is still a very good substrate. It’s just an imitation of model substrates we made and it’s cleaved by RNAse P. Now, if we have a piece of DNA which is made into an
RNA or messenger RNA, whatever you want, and there’s no external
guide sequence involved, you make protein, a regular state of affairs. If you have an extra guide sequence, external guide sequence, hydrogen bonded to the piece of message then cleavage is going to occur at one particular end of the EGS and you’re gonna get mRNA cleavage there by the host cell RNase P which recognizes this as a substrate. And you’re going to
inactivate this particular RNA and you’re gonna stop it from
expressing itself completely. So if the RNA’s a viral RNA or an RNA that codes for
a particular disease, you’re going to absolutely stop it from functioning under those conditions. So that’s the basis of the therapy, that you have this situation and you have the RNase P
which exists inside the cell and so you can do all these things. Now these are some ways of
transporting any piece of RNA. There are various other
mechanisms in which RNA’s involved and I’m not gonna talk
about them at the moment. Let me just say that the
method I’m gonna describe is just as good as siRNA. We’ve tested siRNA on the same targets as we’ve tested the EGS on and the numbers are very comparable. You can have things attached to antibodies which themselves attach to particular parts of cells. You can have them attached
or inserted into viruses which then infect cells. You can have oligonucleotides
encompassed in liposomes which are lipid bodies that
will wrap around a piece of RNA. Or a nanoparticle containing some RNA. Or you can have basic peptides
attached to pieces of RNA. Although our previous work
had been done biologically, that is to say, we made small genes that coded for EGSes and
inserted them into bacteria and they certainly work in E. coli and some other organisms. But now we’re doing these experiments with basic peptides with
morpholino oligonucleotides. And I’ll show you a picture
of that very shortly. And this is just an
indication of the kinds of RNA you could use to shut
down gene expression. There are aptamers which
are small molecules that bind to particular sequences. So these are made out of RNA let us say. Hammerhead ribozymes which
are not very good I think. siRNA which can be used in
human cells for example. Splicing variants which have been used to attack certain diseases in which there is a splicing variant that causes diseases like
Duchenne muscular dystrophy and you can adjust that splicing variants back to the normal situation. In a couple of papers
which have been published you get some improvement
in the muscular dystrophy. The EGS which is, which is what I’m gonna talk about. Locked nuclear RNA which
is another variant on RNA and peptide nuclear RNA. I’m not gonna talk much about these, but they can be useful. This is an old slide showing results with our biological effects. Simply that an E. coli for example, we can inhibit the expression
of various of these genes. When you induce beta-galactosidase
or alkaline phosphatase they’re made at a level of over 1,000 fold of the constitutive level. So normally they’re hardly made at all, but you can induce them and then the level goes up. With one EGS attacking
one site on the message, you knock out 60% of the activity. These are essential genes. But this one is the one
that we’re interested in drug resistance. And we’re looking essentially
at chloramphenicol resistance. And we can stop that, we can reverse that into
chloramphenicol sensitivity of a bacteria 100% by using two EGSes that attack two different sites in the messenger RNA that codes for chloramphenicol resistance. Okay, you can say that’s all right, but conventional antibiotics
are just as good. Well, they’re not just as good. A one-step mutation can convert something that’s sensitive to drug resistance. In our case, three mutations as long as they’re not right
next to each in the EGS can still function. So it’s clear that that is a
much better biological result than the conventional
method of doing things. And this is also has been
replicated by somebody else in actually California, now that I’m here, on amikacin resistance. Just let me go on now. Very briefly I will say that we can now isolate an EGS in a few hours providing you have a supply
of E. coli RNase P on hand which is very easy to arrange, and a 1/2 ml solution of a piece of random RNA. So in our case we might have
something this long here where N is the random RNA in these pieces and we have various
restriction sites here, and we just incubate this. You can label the target RNA, in any particular, any particular RNA you want. You can mix that labeled target RNA with this random oligo here and then incubate it for
15 minutes with RNAse P and run it out on a gel. It takes two hours perhaps. Develop the film on the gel and you will see immediately whether or not you had any cleavage. of the target RNA. And in fact, we’ve shown this prolifically over the last
couple years using this method. We’ve identified EGSes in
many different bacteria and they can be used for drug sensitivity to target pathogenic
regions in pathogenic RNA. It’s a very convenient method. Now let’s get to this new method which is the basic peptide’s attached to morpholino oligos. This is a morpholino oligo. We see the phosphate linkages are not quite what you would expect. There’s a nitrogen in here. This is a morpholino sugar in here, a nitrogen in it. But the bases appear
as they usually appear. And the other aspect of using this is that the morpholinos
are tremendously resistant to nonspecific nucleases. So you can incubate them with human serum or bacterial growth medium and they don’t get degraded
for at least 24 hours, probably much longer than that. And that’s why we use them. Now you attach a basic peptide at one end. And here it’s 14 amino acid residues. R is arginine. X is 6-aminohexanoic acid. So there are four repeats of this. X again and then beta-alanine. And this makes the covalent linkage very powerful in terms
of penetrating bacteria. We published this about a
year and a half ago or so. Let me just say that if we have, if we’re looking at chloramphenicol
resistance in this case and we’re using two EGSes which have been made
into morpholino oligos with a basic peptide attached, these are control
bacteria after four hours. A scrambled oligo of the morpholino, so it’s almost as good as the control. Two separate EGSes, and two EGSes together. So in this particular case, we’re down around 1/1000 of what we would’ve expected for the control bacteria which is actually a
very, very good number. So that’s at 50 micromolar. When we do it with B. subtilis inactivating the gyrA gene in B. subtilis, we only need five micromolar of this. And we’re down to about, in this case, only 0.2
of the viability of this. If we do this at 15 micromolar, we’re down to about 1% of
the viability of the control. So that works extremely well. And let me just say in an experiment that was finished a couple weeks I’ll just show some rough data. We are now doing the experiment with a different basic peptide. It’s a basic peptide that has
been isolated from human cells that controls T cell regulation, and it’s 22 amino acids long. It’s written up here. It is also very basic and we’ve attached that to a morpholino oligo. And this chloramphenicol resistance. So we’re down in the level between 1/1000 and a 1/10,000 viability. Here, these are two
different preparations. This preparation is obviously much better. And that’s what we’re
working on at the moment. This is at five micromolar concentration of the particular
compound that I worked on, that I told you about. So that’s a summary of
some of our recent data. I have some more data on some
molecular biological aspects of the morpholino oligos and how they interact
with sites in message, but I’m not gonna show that to you because it’s getting late. And I’ll just show you
this one other experiment which is an older summary of experiments in eukaryotic cells. I’m sure Fenyong will
recognize some of this. I’m not gonna talk about
cerevisae right now, but we can inhibit flu viruses
in mouse or canine cells by attacking two different
genes in the flu virus. Remember the flu virus has eight genes. And it also has eight
pieces of RNA in its genome. Each piece of RNA codes for a particular one of its genes. And by attacking both of
these particular genes we essentially can
inactivate flu virus by 100%. But I indicate 95% because there’s a 5% error in our methods. These are essential proteins in E. coli, in HeLa cells, I won’t talk
about them more at the moment. But Fenyong Liu has been
working for many years on cytomegalovirus in mice and in humans. Actually these are old data showing that he can inhibit these
particular functions. But I believe that he’s
also been able to show the inhibition of live animals
under these conditions. And also herpes virus which he also did in our lab a long time ago. So I think this is a very promising method to use, and one that’s certainly worth studying. It is not necessarily adding to our molecular
biological knowledge of RNase P itself and what it’s done. But it shows that RNase P can
be used in very useful ways, in this case, to attack
all kinds of disease. And with that I shall leave you. (audience applauds) – [Audience Member] Sid,
I’m curious how the basic peptide morpholino
penetrates into bacteria so efficiently. It’s not the same kind of mechanism that basic peptides like TAT enter into mammalian cells. It must be something different. – I’m not sure it’s very
different at all quite frankly. – [Audience Member] The
TAT peptide penetrates into mammalian cells by
first being internalized into an endosome and from the endosome — – Say the second half
of that sentence again. – [Audience Member] The TAT peptide, basic peptide, penetrates
into mammalian cells by first being internalized
into an endosome. – Okay.
– And then from there into the cytoplasm, that’s not happening. – It’s certainly not that, that’s right. – [Audience Member] I’m curious about the toxicity and pharmacokinetics of these molecules. – I don’t know anything the about it because I haven’t done any
experiments with live animals. However, there are people have done experiments with these kinds of
compounds at AVI BioPharma which is a company now
located outside of Seattle. And they have cured infected
mice under these conditions, and the mice don’t suffer at all under the conditions they used. They just survive for
several weeks afterwards. – [Audience Member] That’s
encouraging, thanks. – [Audience Member] When
you showed the cleavage of some of the polycistronic
messenger RNAs in E. coli, I assume that was an in vitro experiment. – No, we’ve done it both ways. – Because it was–
– For– – [Audience Member] Pretty inefficient. It was only a few percent
of the intact transcript that was converted to product. So how do you expect there– – There are two different slides there. One was of several different operons. That was done in vivo, okay? And then we did the lac operon which was done in vitro and in vivo. – [Audience Member] But
it’s only a few percent of the message even in vitro. Is it more efficient than vivo, the degree of cleavage
of the intact transcript? The full-length transcript persisted. You know 95% of it was still there after you know more than an hour incubation. – Well let me put it this way, with the lac operon, we completely destroy part
of the lac operon in vivo. – Okay?
– So it’s more– – That was with a model system. Now the other, so I don’t know what you
wanna say about that. With the other operons it is true that we don’t have a good idea of the quantitative result
of those experiments. – [Audience Member] Okay. – [Audience Member] So
does the bacterial protein stimulate the RNA of Archaea and so forth. In other words, if you combine the
different proteins and RNAs are the proteins still active? – Which protein? – [Audience Member] The RNAse P protein. So immediately, does the RNAse P protein from E. coli for example, stimulate the RNA of RNAse P from Archaea and so forth? – Well if you’re talking about experiments using an EGS to– – [Audience Member] No,
no, this is going back to the beginning of your talk. – Well just under normal conditions? – Yeah.
– Yeah. The amount of P protein for RNAse P, there are about 400 to
500 copies in the cell. And if you try and activate RNAse P by attacking the message for the P protein which we have done, it actually takes several more hours to kill those bacteria than it does for example, to just change chloramphenicol
resistance to sensitivity because you have to deplete that reservoir until you’re down to maybe
20 molecules per cell which is apparently not enough to keep the molecules going. – [Audience Member] EGS,
what needs to be done to bring EGS as a therapeutic technology in the–
– Somebody has to provide a few million dollars to set up a company to investigate this for commercial purposes and to build a reasonable system for delivering the covalent linkage of the basic peptide to the morpholino. So I don’t know anybody
with a few million dollars who’s willing to do that. And I’m not a great entrepreneur, so I can’t do anything about it. – [Audience Member] I
have a follow-up question. Is it easier to design an EGS for viruses as opposed to cancers? – In concept and in principle, you could for a virus, certainly for viruses. And that’s what Fenyong is doing. – [Audience Member] Well, I wanna follow was Stu’s question. How much we know about the biogenesis of the enzyme in terms of the RNA subunit and the protein subunit in E. coli? – Just a minute, could you stand a little bit further
back from the microphone? Not that far back, okay. – [Audience Member] So much
we know about the biogenesis of the RNA and protein
subunits in E. coli? I mean specifically when the bacteria grow under different conditions
like under stress or under rich medium. Do we see any–
– For the protein? – [Audience Member] As
well as the RNA subunit. – Okay.
– Is there any regulation? – The protein is made in an operon with some ribosomal proteins in it. Supposedly there is a binding, the sequence of the
upstream part of that operon has a binding site for the RNA subunits. But we’ve done a number of experiments in which we tried to regulate the amount of RNA and
the protein in E. coli and none of them have worked. That is to say, changing the amount of RNA doesn’t change the amount of protein. Changing the amount of protein doesn’t change the amount of RNA. That’s about all I can say about that. Is that what you wanted to know? – [Audience Member] Because I’m wondering whether the tRNA synthesis is affected by that?
– Oh, that’s a separate issue. The RNA subunit is under
relaxed stringent control. And I think so is the protein subunit. So if you starve the cells for amino acids or they go into stationary phase, you stop making both the
RNA and the protein subunit. – [Audience Member] Interesting. – [Fenyong] Please join me in thanking Professor Altman for a wonderful lecture today. (audience applauds)
– Thank you. (bright music)

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