COMMEMORATIVE LECTURE
Structural Insights into the Functions of theLarge Ribosomal Subunit, a Major Antibiotic Target
Thomas A. Steitz

Investigator, Howard Hughes Medical Institute and Sterling Professor of Molecular Biophysics and Biochemistry,
Yale University Professor of Chemistry, Yale University, New Haven, CT, USA(Received for publication on Ausust 25, 2007)

Key words: Ribosome structure, RNA, antibiotics, protein synthesis, ribozyme

Chairman (Prof. Yoshikazu Nakamura, Institute for
Medical Science, University of Tokyo): Finally we will
have a lecture by Prof. Thomas A. Steitz. He is the winner of Keio Medical Science Prize. Already, he explainedor talked about his personal history and therefore really I
do not need to repeat the same thing; but briefly I would
like to introduce his career. He graduated in Chemistryfrom Lawrence College,Wisconsin in 1962, and he finished his PhD in biochemistry and molecular biology at
Harvard University. Then he started his professional career path in Harvard University, the same department ofchemistry, and he also spent three years 1967-1970 in
Cambridge, England, where he met so many important
figures as we learned yesterday. Then he got an appointment in Yale University in molecular biophysics and biochemistry in 1970. Since then he has stayed in the samedepartment, and carried up through an assistant professorship, associate professorship and full professorship.
He has received quite many awards. Among those just I
will mention a few important points. In 1990, he becamea member of the National Academy of Sciences in the
United States, and also the same year a member of theAmerican Academy of Arts and Sciences. He received
several awards: for example, in the year 2001, the Rosenstein Award for Distinguished Work in Basic Medical
Science, and the latest one in the year 2004, the Frank
Westheimer Medal of Harvard University. As you know,
this year he received the Keio Medical Science Prize. He
received it; I trust and I hope that this award is the bestand prestigious award. Now we will ask Tom for this lecture about “Structural insights into the functions of thelarge ribosomal subunit, a major antibiotic target.”Prof. Thomas A. Steitz, Department of Molecular
Biophysics and Biochemistry and Department ofChemistry, Yale University, and Howard Hughes
Medical Institute: It is a great pleasure to be here, andthank you so much for that kind introduction. As I said
yesterday, it is always wonderful to come to Japan andvisit. My son and I had a great breakfast this morning; asushi breakfast at the fish dock,*1 the best sushi that I
ever had in my life; it is going to be hard to do this again,
I have to say, back home. My son is out having a goodtime in Tokyo and I am here having to talk, so I am going to have a good time too.
I would like to start out by giving an introduction to theribosome and the ribosome problem, and then provide afew details about how we solved the structure of thelarge ribosomal subunit. Now, I realize that this is an unspecialized audience with respect to structure determination, with one or two exceptions obviously here, but I
want to give a sense of what the problem was that needed
to be solved and how we went about it, but that will only
be five slides, so not to worry. Then I shall talk about
what we have done to understand the mechanism of peptide bond formation. Finally, I shall talk about the antibiotic binding studies that we have done and the examination of the effect of resistance mutations, and finally talkabout what Rib-X Pharmaceuticals, Inc.―the company
that I and some of my colleagues started five years ago―are doing to use this information to design new antibiotics, which looks like it is going to be successful.
I shall start with a fact published in Nature a couple ofyears ago, that in the United States, two million people
will acquire antibiotic resistance infections in US hospitals, and 90,000 will die of it. Indeed, this is a significant

Dr. Steitz is the recepient of the 2006 Keio Medical Science Prize. Presented at the Keio Medical Science Prize Symposium, November 2, 2006.
Reprint requests to: Dr. Tomas A. Steitz 266 Whitney Avenue, Bass 418 P.O. Box 208114 New Haven, CT USA 06520 – 8114
*1 A fish dock at Tsukiji in Tokyo.
1

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problem. Another important point is that the ribosome isa major target of antibiotics―in fact, the major target―
and the large ribosomal subunit is the major target inprotein synthesis. Consequently, our understanding of
how a large ribosomal subunit works and how it is inhibited by antibiotics should indeed to be helpful to attacking this problem.
But let me back up and say a few things about our approach. There are some differences of opinions about
how one should do structural biology. This is my opinion, which was exhibited very well by Dr. Yokoyama acouple of lectures ago. That is: you need to have thestructures of every stage of a process in order to understand that process. It is not just good enough to have one
structure; you need to have them all. So it certainly is notenough to look at the pieces. As you know, most assemblies are made from a lot of pieces. It is easy for us to be
reductionists; we all reductionists, and we are tempted tolook at whatever we can get our hands on, but knowing
the structures of the pieces of a clock does not tell you
how a clock works. But I might say that even if you
know the structure of a clock sitting there, that does nottell you how a clock works either. So what you have to
do is have the structures of the clock or the assembly thatworks at each step in that process. Even though some ofthe steps are metastable, you have to be a clever biochemist and figure out how to stabilize that state so you
can take a snapshot of it, and then you can put it together
into a movie. So that is the philosophy that we have taken with all the problems that we were studying in the lab.
I did show some movies yesterday, and that is how weare looking at the ribosome as well.
We have been looking at all aspects of gene expression,but of course today I am just going to focus on the ribosome and indeed I am only going to focus on the large ribosomal subunit where peptide bond formation occurs.
This schematic drawing of protein synthesis by Jim Watsonshows where our understanding of the ribosome wasin 1964. It was known that the decoding happens in thesmall subunit and peptide bond formation occurs on thelarge subunit. Also shown was the A site, where the aminoacyl-tRNA binds, and the P site, where the peptidyltRNA binds, which he proposed existed, but he did notknow about the E site, where the exiting deacylatedtRNA binds, and he did not know about the polypeptide
exit tunnel, but he did propose translocation. So we will
mention a few things about the tunnel, and we will alsomention about the E site.
The ribosome as I am sure you all know is the largest
RNA assembly that exists. The eukaryotic ribosome is a
bit larger than the prokaryotic ribosome, but otherwise
there are not any larger RNA assemblies. The bacterial
ribosome has 3,000 nucleotides in the large subunit, andSteitz TA : structures of the large ribosomal subunit

another 1,500 in the small subunits; so the whole assembly of the 70S is about two and a half million molecularweight, two-thirds RNA by mass. Think of it as an RNA
machine contaminated with proteins, whereas the spliceosome, I tell my wife,*2 is a protein machine contaminated with RNA. The ribosomes constitute one-fourth of
the bacterial cell mass, so it is very abundant, and ofcourse that is extremely useful if you want to do structural studies, to have a very abundant machine.
Now the early structural work in the mid-70’s wasdone by Jim Lake using negative staining in electron microscopy, and he was able only to show the shapes of thetwo subunits, but his shapes for the individual subunits
are largely right at this resolution of 50 Å or so; then examining the model of the whole ribosome you can seethat the two subunits are sort of snuggling up here like apair with the small subunit lying on top of the large subunit. Using cryo-electron microscopy (cryo-EM), it waspossible to go to somewhat higher resolution by 1997
and position these transfer RNA (tRNA) molecules approximately correctly, showing the CCA going into thelarge subunit and the anticodon loops of decoding a fictionally placed messenger RNA, and then the E-site
tRNA, and more or less over here but again not exactly
positioned correctly. So that is where we were in our understanding of ribosome structures approximately 10
years ago.
I have met up with Venki Ramakrishnan many times―
actually, he was a post-doc at Yale in the 1980s with Don
Engleman and Peter Moore―but I met him in Sweden ata meeting, so we had a photo opportunity, and I am going to use Venki’s structure of the small ribosomal subunit and put it together with our structure of the larger ribosomal subunit to get the structure of the whole 70S ribosome. The two subunits were positioned using the coordinates of the phosphate backbone of the 70S from
Harry Noller and his three A-site, P-site, E-site tRNAs.
Venki, as you may have noticed if you are paying attention to this area, has recently determined at 2.8 angstroms resolution the structure of the 70S assembly with
tRNAs bound in the P site and E site and partially the Asite (5); so there is an even more accurate structure ofthis assembly than shown in Figure 1 but I think for ourpresent purpose you would not be able to tell the difference, nor would I. The ribosomal RNA is shown in whiteand purple for the large subunit and in various colors ofyellow for the small subunit. At the bottom of a large
cleft in the 50S subunit is where we are going to spend a
lot of time, actually, where the peptide bond formation
occurs. There is a hole in the small subunit where themRNA comes in. You can see a very complex folding of
the RNA. Some gaps in between, for sure, but many of thesegaps are filled by the proteins that are fitting in between.

*2 Prof. Joan A. Steitz, Department of Molecular Biophysics and Biochemistry, Yale University , and Howard Hughes Medical Institute.

Keio J Med 2008; 57 (1): 1－ 14

Fig. 1 Space-filling model of the 70S ribosome derived fromthe structures of the Hma large subunit (Ban et al., 2000) andthe T. thermophilus small subunit (Wimberly et al, 2000) docked
using the rRNA from the model (Yusupov et al., 2001) of the 70S
ribosome, and that includes the A-, P-, and E-site tRNAs fromthat model also. The 23S rRNA and 5S rRNA are on the right,
whereas the 16S rRNA is on the left. rProteins are shown. The
A-site tRNA and the P-site tRNA are shown with their 3’ ends
extending into the peptidyl transferase cavity. (Reproduced from“RNA in the Synthesis of protein IN: The RNA World.” PB
Moore, TA Steitz, Cold Spring Harbor Laboratory Press, 2005;
258 Fig.1)

Well, what I would like to first do is use a movie that
Venki Ramakrishnan commissioned to show the overall
process of protein synthesis using both crystal structuresthat are known and electron microscopy reconstructions.
Ultimately I want to say that what we need to have are
the high resolution crystal structures for every one ofthese steps. For the moment we are doing pretty well because we have a lot of electron microscopy images ofthese steps which combined with crystallography provides a pretty good idea of what is happening, but I think
we shall want to get down to the exact chemistry. Thus,
we are going to have to have crystal structures., but thisis where we are at the moment. This movie comes from
the Medical Research Council (MRC) laboratory. First,
the small subunit binds to the messenger RNA and is facilitated by initiation factors, IF2, IF1 and IF3 , and they
then position the formyl-methionine-tRNA (fMet), theinitiator tRNA. Then that assembly meets up with thelarge subunit, and the factors dissociate leaving the fMettRNA positioned in the P site. Now elongation begins
with the delivery by EF-Tu of an aminoacyl-tRNA, GTP
gets hydrolyzed and then you get a tRNA accommodation. There will be a little “zing” here as the peptide

3bond is formed; then it will go to what is called the hybrid state, which moves the CCA ends over one step into
the E site and P site. Now EF-G comes along, hydrolyzes
GTP, translocates both tRNAs and then another tRNA isdelivered by EF-Tu, and this process goes on at a fasterrate in the movie. You see now polypeptide coming out
the tunnel; there is a tunnel that goes about 100 angstroms through the middle of the large subunit, and thefolding of the polypeptide happens on the other side of
the tunnel. Actually, another interesting process is the
cotranslational insertion of polypeptides into the membranes which we do not know anything about yet in detail. Then the factor comes along to stimulate the hydrolysis of the peptidyl-tRNA which then releases the protein, and now a couple of proteins come along to clean
up the job of removing the message and dissociating thecomplexes. So that is the whole process, and now we aregoing to focus our attention on the large subunit.
I should mention that my work on the ribosome wasinitiated when Nenad Ban joined my laboratory in 1995.
I had certainly considered initiating this project for along time; Ada Yonath’s lab had been pursueing the ribosome for a few decades and many of us noted that while
she had been successful at growing some crystals, thesuccess at solving the structure had not been so noteworthy. So we thought we should have a hand at it as well,and I thought Nenad Ban was the right guy, and indeed
he was. He came in and he grew the crystals repeating
the crystallization, I might say, of Ada Yonath, and thenmade heavy atom derivatives of the critical kind. I am
going to come back to what he did that was so good.
Then he was joined two years later by Poul Nissen. Poul
Nissen came from Aarhus and he has gone back to Aarhus; he figured out how to make fatter crystals with fewer of the kinds of problems that the earlier crystals had.
He worked with Nenad Ban to solve the structure. So really the structure was solved by these two wonderful
post-docs, with occasional help from a few other rotating
graduate students and another postdoc in the lab.
What were the crystallographic problems? Here are myfive slides, but I want to give you an indication of why it
was that it was difficult to solve the ribosome. I must saythat from the bottom of Mt. Everest, which is what theribosome structure determination was considered, its
structure determination looked very daunting. Now ofcourse, every time you get to the top of the mountain,
you say, “Well, gee, that was not such a bad thing, It really looks pretty easy.” But at the bottom, it looked bad,and why was that? Well, the crystals initially were kindof thin, they had a number of problems and they were
twinned. We could get very good diffraction, but Poul
Nissen figured our how to make fat, well-behaved crystals. So that was a great start. The other problem, in fact
the major problem-solving novel protein structures fromthe days of myoglobin on, is making what is called a4

Steitz TA : structures of the large ribosomal subunit

Fig. 2 The appearance of the large ribosomal subunit from H. marismortui in electron density maps at different resolutions. The
subunit is shown in the crown view at (a) 20 Å resolution, (b) 9 Å resolution (Ban et al., 1998), (c) 5 Å resolution (Ban et al., 1999),(d) 2.4 Å resolution (Ban et al., 2000). CP designates the central protuberance. The L1 stalk, which is visible at low resolution,
disappears as resolution improves.

heavy atom derivative of the crystalline complex and locating that heavy atom. How do you do that in this case?
It is a very large assembly; it is one and a half million
molecular weight, and if you add just a single uranium
atom, even though you think of uranium as being big, it
is very, very small compared to the ribosome. So what
was done was to use a very large heavy atom clustercompound. The compound, containing 18 tungstens plus
a few other atoms, has 2,000 electrons, notably larger
than uranium. Now why is the cluster compound useful?
It is useful because at low resolution, that is, at a small
diffraction angle, not the angle that is going to tell you
where all the atoms in the molecule are, this cluster scatters as one assembly. You get the square of the numberof electrons as your signal. Squaring 2,000 rather than 76
is a big thing. So if you look at the intensity of the scatter as a function of resolution: at 12.5 Å resolution,
which is almost electron microscopy resolution, youknow, low resolution―and look what is happening to thescatter for these cluster compounds, the intensity of thescatter goes through the roof. That means we have a verystrong signal from the heavy atom cluster compound.
Thus, we calculated the first maps at 12 Å resolution.
Now, most of you will not know what a difference Patterson map is and I will not try to tell you what it is, but
it is what we look at to locate the positions of heavy at-oms, and we have to see a peak. You can recognize apeak, which there is, and these peaks in different projections have to be correlated in a particular way, which
they are. So when Nenad Ban calculated this, we could
all say “Eureka! We know this is going to work.” Indeed,it did.
Figure 2 shows the progress in structural studies of thelarge ribosomal subunit. In 1996 there was electron microscopy at 20 Å resolution. I always say this E.M. image looks like a Henry Moore statue here; it looks like aman on his knees holding some sort of a torch with hishead here. In any case, there are a lot of holes in it. In
1998, we could see in the first 9 Å resolution x-ray map
what looks like RNA helices, so this was the first timethat one could see recognizable structural features in anelectron-density map of the ribosome. Now in 1998, wepublished how we were doing business, 1 and then awhole lot of other groups started doing exactly the samething, quite reasonably, and by 1999, there were several
simultaneous or almost simultaneous publications of
5 Å, 6 Å or 7 Å resolution maps and at that resolution
we could fit in little pieces of known ribosomal protein
structures, which was nice. In 2000 we got a 2.4 Å resolution map and we could fit all 100,000 atoms. So then
we knew the atomic structure of the large ribosomal subunit.2

Keio J Med 2008; 57 (1): 1－ 14

This representation of the structure of the 50S subunitthat was published in 2000,2 shows the RNA backbone
in orange and bases in white fairly tightly packed aroundthe active center and the proteins embedded around thesurface (Fig. 3). I am not going to go in to detailed discussion of what we learned about the structure, but there
was a lot that we were able to understand about principles of RNA folding and RNA tertiary structure interactions and how in the world a large molecular assembly ismade out of RNA. But I will not talk about that today.
What we are going to do is we are going to split the subunit in half so we can look in the inside; splitting it likean apple and opening it up. In white are those RNA regions that are in the interior that has been split and ingreen the protein penetrating into the interior helping topack the interior and hold it together. There is the peptidyl transferase center where we are going to spend mostof the rest of the time, and there is that exit tunnel,
through which the newly synthesized polypeptide goes
and then folds outside the far end; it is about 20 Å in diameter in some places and it is 10 Å in others and it isabout 100 Å long. All the antibiotics we are going to talkabout, by the way, bind in the tunnel either in the region
below the peptidyl transferase center or a little bit closer
to the site of the peptide bond formation.
The active site is composed entirely of tightly-packed
RNA, and I might say is very conserved. Fortunately, it
is not 100% conserved, otherwise antibiotics would notwork as therapeutics, but it is very conserved. Shown in
red are all the rRNA nucleotides that are conserved, very
conserved, and if you consider that eubacteria, archaebacteria, eukaryote…We are talking about all of evolution; that is, the conservation is very high. There is where
the substrate binds. If we ask “At the point where peptide bond formation occurs, what is the closest protein?”,the answer in 2000 in the Hma 50S subunit was “nothing
very close at all”; now one can see in a eubacterial species, there is a protein, L27, that comes a little closer, but
not much. So we were able to conclude that ribosome isa ribozyme; that had been speculated over the years because how else could you have a machine to make a protein if you did not have a protein? It is the chicken andthe egg problem; which came first? The answer is: RNA.
What I would like to turn my attention to now, however, is how can we understand how an RNA is able to catalyze the chemical reaction of peptide bond synthesis?
What is it about the RNA that allows that to happen?
This is the work primarily of Martin Schmeing, however,I might say, Jeff Hanson helped a bit in the early stages.
So here is a picture of Martin, the graduate student―or
was; he is now a post-doc with Venki Ramakrishnan
helping Venki conquer the world; Martin is very good―and he is shown next to his sailboat that is parked on ourbeach in Connecticut; I tried to get him to stay a little bit
longer, so I said “You could keep your boat on our

5Fig. 3 A space-filling model of the large ribosomal subunit from
H. marismortui with a transition state analog bound viewed down
the active site cleft (Nissen et al., 2000). Bases are white, thesugar-phosphate backbone is grey and the substrate analog (in thecenter) is dark. Proteins whose structures are defined by the 2.4 Å
resolution map are black. Grey ribbon represents proteins L1 andL11, whose structures are independently known that have been
positioned approximately using lower resolution electron density
maps. Identification numbers are provided for all proteins, andCP designates the central protuberance. (Reprodced from “The
Structural basis of ribosome activity in peptide bond synthesis.”
Nissen et al., Science 2000; 289: 920 Fig.4)

beach,” and then winter came and he went off to Venki.
Martin is a wonderful person, and as I mentioned yesterday, his mother was a roommate of my wife’s and I wasbest man at his father’s wedding, and I did not meet him
until he walked into my laboratory. It was wonderful.
What is the reaction that is catalyzed? You are alltaught this in introductory biochemistry. The alpha amino group of the aminoacyl-tRNA attacks the carbonyl
carbon of the peptidyl-tRNA. It forms an oxyanion containing tetrahedral carbon intermediate, and then it
breaks down to give products with the peptide beingtransferred to the A-site tRNA.
We are always optimists, so we of course did try to diffuse aminoacyl-tRNA into our crystals, even though we
knew this was lunacy and it would not work, and ofcourse it did not. So what we actually have done is wehave used small substrate analogues―CCA-amino acidor CA-amino acid or CCA―peptide.We have looked atvarious combinations of all these complex structures. We
have many analogues of the transition states that wehave looked at and also of structures of the products. I
will show you a few of these structures, but I am not going to show you anything like the large number that wehave determined. Again, if we cut the ribosome in half

6Fig. 4 The structure of the large ribosomal subunit from H.
marismortui with products of the fragment reaction (CCAand C-pmn-pcb) bound in the peptidyl transferase center. A
space-filling representation of the complex with three intact
tRNAs added in the positions tRNAs assume when bound to
the A, P, and E sites of the 70S ribosome (Schmeing et al.,2002). TRNA is white, and the ribosomal proteins are grey. The
subunit, which is oriented in the crown view, has been cut in halfalong a plane that passes through the peptide exit tunnel, and thefront of the structure has been removed to exposed the tunnel
lumen. The active site area is in a box. A close-up of the active
site showing the peptidyl product (CC-pmn-pcb) bound to the
A-loop. (Reproduced from “A pre-translocational intermediatein protein synthesis observed in crystals of enlymatically active
50S subunits.” Schmeng et al., Nature Structural & Molecular
Biology 2002; 9: 225–230 Fig.4a)so that we expose the polypeptide exit tunnel―you can
see the proteins that come in and contact the tunnel (Figure 4). Here are the positions of the three tRNAs andthis is where we are going to look at the substrate analogues that we have found. As I often point out, for the
rest of the lecture, even though this is, I think pretty good
research, we are going to think inside the box that surrounds the PTC rather than outside the box.
This is where we were in our understanding of substrate binding to the PTC3 slightly after 2000. We knew
from Harry Noller’s work approximately where the twotRNAs are positioned, and of course the two tRNAs areoriented parallel to each other, but their CCA*3 ends arerotated relative to each other by 180 degrees. This is the
CCA of the P-site substrate, and C74 and C75 are making two base pairs with the ribosomal P loop, and the
A-site substrate makes one base pair with the A loop. It
is rotated by 180 degrees, so that the alpha amino groupof the A side substrate can be close to the carbonyl carbon of the P-site substrate. Now, we thought this looked
pretty good, sort of, except that we realized that this alpha amino group was not positioned quite right for the

Steitz TA : structures of the large ribosomal subunit

nucleophilic attack, but we sort of ignored and said,“Well, maybe it is accuracy,” but it actually had something to do with the way we did our experiment.
Well, I went to Sweden a couple of years ago, and after
my lecture, somebody asked this question: why is the
peptidyl-tRNA not hydrolyzed in the absence of the
A-site substrate? Now, since the rate-limiting step in
peptide bond formation is the delivery of the aminoacyltRNA, the A site is mostly empty. If the enzyme is set up
with the correct structure to catalyze the nucleophilic attack, why can it not activate water molecules, which actually it does in the final step of deacylation of the polypeptide? Well, I did not know what the answer, so I said,“I do not know what the answer is.” I went back, andMartin Schmeing had already worked it out while I was
having fun in Sweden. It turns out that the answer is thatwhen the correct A-site substrate binds, it rearranges the
structure of the active site in such a way that now the ester-linked peptidyl group gets correctly oriented and gets
exposed.4 It is buried in the absence of a correct A site
substrate.
If you look at the position of the peptidyl group, when
you have just the peptidyl- CCA bound, and nothing in
the A site, this peptidyl group is protected on one side bya U base, at the position that a water molecule will haveto take in order to attack. It cannot get there because it issterically excluded. Similarly, on this side it is sterically
excluded by some other RNA. So the enzyme ribosome
is very clever. It is protecting its substrate in the absenceof the A site substrate.
This is a complicated slide (Figure 5), but I will show
you a movie shortly, which I think will clarify any questions you might have. What we were able to do, or what
Martin Schmeing was able to do is look at one substratethat does not induce the change, and that is shown on
this lighter color here, and the bases of the ribosome areshown also in lighter color. When that shorter substrateis bound, or in the absence of this substrate at all, this U,
as I had shown in the previous slide, is maintaining theposition of the peptide in this orientation and burying it
and excluding water. However, if you add CCA, or if
you have an intermediate complex which is shown inblack, CCA, there is a conformational change in the ribosome, which is interesting and finally results in themoving of this base out of the way so that this peptide
can reorient and you can get a nucleophilic attack. It isreoriented for the attack by the alpha amino group. Let
me show a movie of the induced conformational change.
So we start out with the peptidyl group protected: you
see this U base moving out of the way. The PTC is goingto the induced structure. Now the alpha amino is in a position so that it can attack, which it does, and then you
get the intermediate. That is what happens. Now this is*3 The universal CCA trinucleotide common to all tRNAs located at their 3′-ends is shown to play an important role in peptidyl transfer reaction.

Keio J Med 2008; 57 (1): 1－ 14

7

Fig. 5 Movements of rRNA and peptidyl-rTNA are induced by proper binding of the A-site substrate (Schmeing et al., 2005a). (a) Aview of the A-site with three different substrate analogs bound. Without C74, ChPmh and rRNA colored lighter is positioned higher
in the A-site. In substrates with C74, both CChPmn (darker rRNA) and the transition state analogue (TSA ) (black rRNA), C74 stacks
with rRNA base U2590 (2555), shifting the substrates down and the α-amino group closer to the ester carbon of the P-site substrate,
rRNA base G2618 (2583) shifts to maintain the A-minor interaction, causing methylU2619 (2584) to also move. (b) View of the
P-site of the same three complexes. The movement induced in G2619 (2583) by A-site substrate binding breaks its G-U wobble pairwith U2451 (2506), which swings 90˚. MethylU2619 (2584) and U2620 (2585) also shift to allow the ester group to move from theposition it occupies when CCApcb (light) is bound together with ChPmn, to that when CCApcb (medium) is bound with CChPmn, andfinally to that when it has been attacked by the A-site substrate, as shown with the TSA. (Reproduced from “An Induced-fit mechanism
to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA.” Schmeing et al., Nature 2005; 438: 520–524 Fig.3)

the proper structure for a pre-reaction ground state (Figure 6). So this is the cast of characters and where they
are when chemistry is about to happen. So what is happening? There is the attacking alpha amino group, and it
is interacting with the 2’ hydroxyl of the P side substrate,and it also interacting with A 2451, A2486 in the Haloarcula marismortui numbering.
Well, why is that useful for catalysis of peptide bondformation? The first thing that any enzyme will do is tocorrectly orient the substrates, so they are ready for attack, and that is probably the biggest energetic factor toanything in catalysis. Jenks said this in 1971: entropy orloss of entropy by orienting the substrate is the biggest
factor in catalysis. So that is important.
But what else is happening? Are these functionalgroups activating that alpha amino group? Well, we hadsuggested that maybe this A was doing something, butthat was soundly shown to be incorrect, most effectively
by Rachel Green,5 who showed that if you used the fulltRNA in the A side, you can mutate that A to any of thethree other bases, and it does not make any difference topeptide bond formation. Consequently, that A is not involved in catalysis. Well, there is only one thing left, andthat is the 2’ hydroxyl group of the P-site A76. Scott
Strobel’s lab showed 6 that if you make that A76 sugar a 2’
dioxy, and get rid of that P-site 2’ hydroxyl, the rate of
peptide bond formation goes down by one million fold;that is a big number. I will come back to this; I think that
this large decrease is not entirely what the catalytic enhancement is relative to non-catalytic but it is a big effect.
Some earlier work by Andrea Barta in Vienna7 using asmall peptidyl-AMP substrate has seen a similar but notsuch a large effect, and she proposed the following

8Fig. 6 Pre-attack conformation of the substrates of the peptidyltransferase reaction. The hydroxyl group representing the α-amino group of the A-site substrate, CCh-Pmn, is in position to
attack the ester group of the P-site substrate CCApcp. It is withinhydrogen-bonding distance of the N3 of A2486 (2451), and the2’-hydroxyl group of the P-site substrate. In the ground state,
the two reactive groups are 3.7 Å apart (Schmeing et al., 2005a).
(Reproduced from “An induced-fit mechanism to promote peptide
bond formation and exclude hydrolysis of peptidyl-tRNA.”
Scheming et al., Nature 2005; 438: 520–524 Fig.4)

mechanism. Why is this two 2’ hydroxyl so effective?
Well, it picks up a proton from the attacking alpha aminogroup, and donates a proton to the leaving three 2’ hydroxyl (Figure 7). It turns out that this P-site 2’ hydroxyl
is completely buried, so water cannot get there any otherway. Anyhow, I think this proton shuttle has a big effect.
Then we can ask, is the transition state being stabilized?
That is the other thing that enzymes do. We have a nicecomplex, and we can see a water molecule which is positioned by the ribosome,8 and it could be the water molecule which is polarized is also contributing, but it ishard to estimate how much. So in the end we conclude
that catalysis is accomplished by substrate orientation,
the proton shuttle of the 2’ hydroxyl and maybe somestabilization of the transition state by a water molecule.
This then summarizes how catalysis occurs at the PTC.
I think we basically understand it at this point, and it fitswith a lot of kinetic and modification data by others. So
let me just wind up with a movie. Just to put this all together: this is a movie made by Martin Schmeing.
We are moving into the peptidyl transferase center. I
can still talk over all this music. Here is the P loop andhere is the A loop. That is the P-site substrate coming in,
making these two C-G base pairs, and it is buried, the ester-linked peptide is protected. Here is the attacking
A-site substrate coming in. It is making hydrogen bonds

Steitz TA : structures of the large ribosomal subunit

Fig. 7 A schematic drawing of the proposed “proton shuttle”
model for peptide bond formation (Dorner et al.,
al ., 2005).
The P-site peptidyl-A76 is being attacked by the A-siteaminoacly-tRNA. Protons are shown shuttling from the α-aminogroup to the 2’ OH and from the 2’OH to the 3’ hydroxyl group.
(Reproduced from “Molecular aspects of the ribosomal peptidyltransferase” Doner et al., Biochem Soc Trans 2002; 30: 1131–6
Fig.3)

with the A-loop being oriented, and you can see that it is
not well oriented for attack, but now if we add another C
to form CCA, you get the activated form. Now we areready for the attack of the alpha amino group on the carbonyl carbon. Then we get the transition state. It is stabilized by the water molecule. Then it breaks down. Then
we have the products. Now the deacylated CCA will then
go to the E site, the exit site. Martin Schmeing determined the structure of an analogue of an E-site substrate―actually, the whole tRNA goes, of course, but we do
not know where the rest of it is, for the moment. Anyhow, this lonely CCA is wandering over to a binding sitethat exists for the CCA. I might say this binding site isdifferent between eukaryotes and prokaryotes and affects
what kind of antibiotics will bind here. I am not going totalk about that. The reason that the amino-acylated tRNAcannot bind is because there is no room to accommodate
the amino acid. Now as the peptidyl-CCA travels fromthe A-site to the P-site you can see this 180 degree rotation, going from the A site to the P site; the peptidyl CCA
has to rotate 180 degrees, and now one is ready to start
all over again. That is the reaction.
So now let me turn to the antibiotic studies for the last
15 minutes or so. These were done largely by Jeff Hanson initially. He started off by looking at erythromycin,
but it did not bind. That is because it turns out that archaea are eukaryotes in disguise in many ways. That is,
many of the binding sites for antibiotics in this Haloarcula marismortui look more like the mammalian bindingsites than they look like the eubacterials. Consequently,

Keio J Med 2008; 57 (1): 1－ 14

9

Fig. 8 The interaction of macrolide antibiotics with the large ribosomal subunit of H. marismortui (Hansen et al, 2002b). A
superposition of several macrolides/large ribosomal subunit complex structures. Structures containing carbomycin, tylosin,
spiramycin, and azithromycin have been superimposed by aligning corresponding 23S rRNA atoms in four independently determined
crystal structures. The macrolide rings of the four antibiotics bind to virtually the same site in the proximal portion of the peptide exit
tunnel. In the case of the 16-membered macrolides examined (tylosin, carbomycin, and spiramycin) A2103 (2062) swings down sothat its N6 can form a covalent bond with their aldehyde substituents. (Reproduced from “The Structures of Four Macrolide Antibiotics
Bound to the Large Ribosomal Subunit.” Hansen et al., Molecular Cell 2002; 10: 117–128 Fig.5)

he could not make a complex with it. But he could lookat other macrolides that bound more tightly. So for example, erythromycin, which was a 14-membered ring,
did not bind; but azithromycin which is a drug made by
Pfizer, selling for a billion and a half dollars or so a year,
did bind, and tylosin, which has a 16-membered macrolide ring, did bind. Most of these others in the MLSK
class did not bind, however. But the 15-and 16-membered macrolides did bind, and they bound in the tunnel.
If we superimpose more or less where they all are, you
can see that they are roughly binding the same place(Fig. 8); they each have different sugar substituents, andthese sugars are reaching out and touching the bases. Allof the interactions are with RNA. There is some little interaction here with L22, with tylosin, but otherwise all
the interactions are with the RNA. Some of them haveextensions that go up towards the peptide bond-forming
regions, the peptidyl transferase center, and we saw a covalent link formed between these 16-membered macrolides and this A.
How, then, do macrolides work to inhibit protein synthesis? Here is where they are located.The binding site
for the macrolides is in this tunnel, and if we look up thetunnel here, this is what it looks like. There is the peptidebond-forming region. It is quite empty, looking up thetunnel, and then here is carbomycin bound partly blocking the tunnel. It is blocking the egress of the polypeptide. I call it molecular constipation; it just stops thepolypeptide from coming out.
We wanted to look at the effect of mutations on the resistance. Since we have, so to speak, the resistant strain,that is we have the one that looks like the eukaryote, we
wanted to go backwards and make it look like the eubacteria. What we have is a G in this position. If you mutate
A2058 to a G in eubacterial ribosomes, that reduces thebinding constant for erythromycin by 10,000 fold. Sothat is why it does not bind to our subunit since we havea G in this region.
All those antibiotics that I showed you on the previous
slide of the MLSK antibiotic, mostly do not bind because
this is a G. Why is that G a problem? Well, if you look at
the one that does bind, the 16-membered macrolide, thering is right above this two-amino group of the G. We
posited that that is getting desolvated―energetically unfavorable―and the packing is not quite as tight, because
the macrolide ring has to back off a little bit due to thisamino group end poking into its center. So we thought,
what if we changed this G back to an A? Consequently,
Daqi Tu, a joint graduate student of Peter Moore andmyself, and Gregor Blaha, a post-doc in my lab, did alittle genetics on Haloarcula marismortui; not a wonderful genetics system, I have to say. They mutated that G
to an A, and then said, “What happens?” Well, it turns
out all the macrolides, lincosamides, streptogramins, andketolides (MLSK) antibiotics bind just fine.9 So here
they are. We were not able to bind clindomycin before,

10

Fig. 9 Macrolide interactions with the large ribosomal subunit of
H. marismortui. Comparison of the location and conformationof azithromycin bound to the wild-type H. marismortui large
ribosomal subunits, and G2099A mutant large ribosomal
subunits from the same organism (Tu et al., 2005). Structuresof both forms of the subunit with the drug bound have beensuperimposed. (Reproduced from “Stractures of MLSBK
antibiotics bound to mutated large ribosomal subunits provide a
structural explanation for resistance.” Tu et al., Cell 2005; 121:
257–270 Fig.2F)

but it binds fine to the G2058A mutant ribosome; virginiamycin S and M bind, and so on, and erythromycinbinds wonderfully. If you compare what we were able todo when we had the wild-type subunit in three millimolar G2058A mutant erythromycin, it did not bind. However, at 0.003 millimolar, when we have only 33%―wehave now made it 100% mutant subunit, but we could
even do it with 33% of the crystal being this―it binds
just fine. So we figured that we have changed the affinity
by 10,000-fold, that is with just that one change, we havecompletely reversed the affinity. What happens withazithromycin? We can bind azithromycin to wild-type
and mutant Haloarcula marismortui large subunit ; itbinds almost exactly the same way as when it was boundto the mutant; it is just raised up maybe an angstrom or afraction of an angstrom because of the presence or absence of this N2 group of the G. (Figure 9.)This result says a couple of things about our antibiotic
studies. One of the things it says is that our complexes
with this Haloarcula marismortui 50S subunit are goodfor drug design, because even if the affinity is down, theway in which the substrate binds is largely the same.
There has been some work on antibiotic complexes done
by Ada Yonath, but I have taken all of those comparison
slides out. I think most of her work that can be com-

Steitz TA : structures of the large ribosomal subunit

Fig. 10 The positions of seven antibiotics and A-site plus P-site
substrates bound to the peptidyl transferase center (Hansen
et al., 2003). The ribosome has been split open to reveal the
lumen of the exit tunnel and adjacent regions of the peptidyltransferase site. Ribosomal components are depicted as acontinuous surface at two positions where splayed-out bases
provide hydrophobic binding sites for small molecules. Seven
independently determined cocrystal structures have been aligned
by superimposing the 23S rRNA in each complex. (Reproduced
from “RNA in the synthesis of protein IN: The RNA world.” PB
Moore, TA Steitz, Cold Spring Harbor Laboratory Press, 2005;
P271 Fig.6)

pared with what we have done on macrolide binding is
simply not correct because of the lower resolution of
their crystal diffraction. We have been able to find alarge number of different antibiotics that bind, and I am
not going to go through them all for want of time. We
have looked at lots of them. I just want to point out thatthey bind the different but adjacent sites (Figure 10). So
here is place where peptide bond formation occurs. We
can bind anisomycin, puromycin, virginiamycin, blastociden, chloramphenical―actually, Ada Yonath’s groupsees it correctly up here in the PTC―and carbomycin.
We also have many other antibiotic complexes. So thepoint is that these different chemical types are binding todifferent but close places, so what you can do to create a
new drug is tie them together.
I am going to talk now for the last five minutes aboutsome work that comes entirely from Rib-X Pharmaceuticals but was enabled initially by the structures of these
antibiotic complexes. This is the company that I andsome of my colleagues started about five years ago.
They have been able to develop a strategy that seems towork in making potential antibiotics. What they are do-Keio J Med 2008; 57 (1): 1－ 14

ing is taking the antibiotics that bind nearby but to different sites, and chemically tying a part of one to a partof another; so you can take a macrolide and tie it tosomething that is nearby to each other or you can takeone of these and tie it to another one. That is what they
are doing. I am going to give you one example. The example is taking sparsomycin which actually does notdistinguish between eukaryotes and eubacteria, and linezolid which is a relatively new antibiotic, very effective,
and that is shown right here. They partly overlap at thisposition. So what is done is to chemically bridge these
two molecules, and then modify them a lot. Of course,
one has to make some modifications so that you do nothave the properties of sparsomycin, namely that of working against eukaryotes as well as prokaryotes. This slide
shows that you can make a compound that is selective
against eubacteria and discriminates against eukaryotes,
and is beginning to get some compounds with goodbinding constants. Then what is done is use a combination of computational chemistry―that is, to decide what
would fit, what seems to have the right properties, chemical properties, and synthesize a bunch of additional
compounds. They go through cycles of compounds―in
this case, showing a couple of hundred ones―and thendo some microbiology to see how the inhibition or translation is going for prokaryotes versus eukaryotes. Now
at the end of the cycles of design and synthesis they get acompound that exhibits very nice selectivity between eukaryotes and eubacteria. Then you look at their effectiveness against some tough strains, and you look at the minimum inhibitory concentrations in micrograms per mil,(if it is below four it is okay). Thus, 0.25 is excellent,
two is fine, so they are getting compounds that are effective against two strains that are resistant to other antibiotics. That is just an example.
Then you can look at two compounds. The affect of the
Rib-X compound against various kinds of staphylococcus pneumonii, haemophilus influenza, and a couple ofvancomycin- and linezolid-resistant strains is comparedwith that of azithromycin, a macrolide, sold by Pfizer forquite a bit of money. You can see big MIC numbers like
128, in the case of azithromycin, while all the numbers
for the Rib-X compound are below one, a big difference.
They have been able to make some compounds that atleast in the microbiology assays are very good. They
have now taken one compound into phase one clinical
trials. I have been traveling recently, so I have not paid
attention to what is happening in the last few weeks, but
the last I heard they were about three-quarters of the way
through phase one and I hope it is going to work. In
phase one you are checking for toxicity, bad side effects.
They hope to get this compound into phase two shortly.
They have a different category of potential antibiotic
made by combining a macrolide with another functionalgroup. They like to call it azithromycin-plus, because it11

is taking azithromycin and trying to make it even better.
That they hoped to put in phase one shortly. So the structures I would say are proving to be useful to design new
antibiotics.
I want to do two more things before I stop. One is I
want to mention people whom I have not mentioned already who have worked on this structure. These include
Dan Klein, in my lab, who refined the structure, Betty
Freeborn and Larysa Vasylenko, technicians in Peter
Moore’s lab who made the 50S ribosome for us, andKevin Wong in Scott Strobel’s lab who made most of thesubstrate analogue compounds that we used.
Finally, without going into any detail―what are wedoing now? Why do we not have any new structures toshow right away? That is because we are trying to growcrystals of the ribosome captured in all the states. We aretrying to obtain crystals of 70S ribosomes with EF-G
that diffract to higher than 7 Å resolution. We have recently gotten some crystals of 70S complexed with
EF-G. I do not know to how high resolution it is going todiffract ultimately. We want 70S with EF-Tu, captured
delivering the aa-tRNA and ask: how does the ribosome
tell the difference between the correct tRNA and thewrong one, so that it knows to hydrolyze the GTP, andhow does it know when to hydrolyze the GTP, after thecodon has been correctly recognized by the tRNA anticodon 70 Å away? How do you do that? We have tohave some structures of ribosome complexes with factors. We want to look at the release factor complex. Venki Ramakrishnan has one at 5 Å resolution, but we still
do not know what the factor is doing to stimulate the hydrolysis of that peptide bond; that process is chemistry,
we do not know its structural basis yet. Then we want tounderstand how the ribosome makes polypeptides go
through the translocon. A structure of the translocon has
been determined, and there are some reasonable electron
microscope (EM) pictures of the ribosome bound to thetranslocon, but I want to know how does the translocon
know when to open up so that a membrane protein can
go into the membrane? How does any polypeptide sequence to go through this channel without its leaking
protons? How does that happen? All of us can make upnice stories, but I want to know the answers. So we areworking on all of those questions. The other problem wewant to work on is the structure of eukaryotic ribosomes,
because eukaryotic protein synthesis is different fromprokaryotes in the initiation phase. Particularly the small
ribosomal subunit differences are of great interest, andwe want to ultimately get structures of the small subunitwith initiation factors to understand how this process
happens.
Last year, two really good people in the lab got somecrystals, and I labeled this slide as “crystals of yeast 40S
ribosomal subunit”. They had obtained crystals of material that sediments 40S. Some of them diffracted very

12poorly and some of them diffracted to about 3.8 Å resolution. After a while, Ivan Lomakin, for reasons I do not
know, ran an SDS gel of one of these crystals and got
two bands, one at 210,000 molecular weight and the other one at 220,000 molecular weight. So they solved thestructure, and it does not look much like ribosome. Itturns out it is yeast fatty acid synthase, which sediments
at 40S. So what they were doing was purifying their 40S
ribosomal subunit by crystallizing yeast fatty acid sythase which is a hetero hexamer of two and a half million
molecular weight, and I have to say, it is a very fascinating structure. It has eight different active sites in it andall the chemistry of fatty acid synthesis is happening in
the inside of this large assembly. Although it can be agreat distraction from our ribosome work, we shall actually try to understand how this magnificent assembly
works.
So science is full of surprises, but I think I shall stop
here, and say thank you once again very much for my invitation to come.
Chairman (Prof. Yoshikazu Nakamura, Institute for
Medical Science, University of Tokyo): Thank you very
much, Tom. An extremely wonderful lecture on ribosome. It is a really attractive and great achievement ofstructure solutions. So probably he will accept questions
or discussion. It is open to questions. Yes, Mr. Yokoyama, dir.
Prof. Shigeyuki Yokoyama (University of Tokyo, RIKEN Genome Research Center): [Inaudible] necessary,
because of the rigidity of the ribosome structure?
Prof. Steitz: How much have we considered the possibility that new drugs will change the structure? In theideal world, of course, you would like to design a drugthat does not do that, because of course as you know,changing the structure, unless the two structures are very
close in energy, is going to cost energy and that will reduce the binding constants of the compound. So I thinknature, as far as I can tell, seems to have evolved inhibitors that produce not many changes, and so the antibiotics that we have bound by and large do not change thestructure very much at all. The only ones that change arebases that we can sometimes see a couple of positions
even in the absence of the inhibitors so that the energy
costs of moving it is very small. So I think our goal is totarget the structure that we see so that we do not have to
pay the energy penalty of changing the structure.
Chairman: Any other questions? Yes.
Unidentified speaker: I liked your video very much,and that was an assembly of snapshots. I am wondering
what is the real speed relative to this. Do you have acomment on that?
Prof. Steitz: It is faster. I do not know; it is many ordersof magnitude, though. I think it is about 50-100 peptide
bonds form per second, so it is much faster than the

Steitz TA : structures of the large ribosomal subunit

movie which forms one peptide bond in about 3 minutes.
Chairman: Any other questions? So Tom, you have now
the new compound antibiotics candidate, and what
would be the frequency of the resistant one appearing
against it?
Prof. Steitz: Well, as I like to say in the United States,
where as you know intelligent design is one of those
things that comes up in discussion of the origin of theuniverse, I always say that evolution trumps intelligent
design, and that is true with antibiotics and I think it isalso true with how we arose. It is clear that the ribosome
will continue to evolve, and will continue to get aroundthose pesky antibiotics. I suppose if you are in the pharmaceutical business, you can consider this a plus, because you know you will always have a job to do, but I
do not see any way in which one can come up with anantibiotic in which you are never going to have to deal
with this problem. I think you just have to have continued creativity at a faster rate than the bugs.
Chairman: Yes, definitely we need new compounds.
Any other questions? Yes.
Prof. Hideyuki Okano (Department of Physiology,
Keio University School of Medicine): You made comments about the future direction. I was very interested inthat case. So is the general idea that as regards eukaryotic ribosome there is some membrane-bound and pre-ribosome, and it is likely that membrane-bound is forming
a complex translocon, as you call it. Is it some induced
fitting structural differences between the membranebound and pre-ribosomes? Because they are very different in the function. So in the case of membrane-bound,the peptide should go into the endoplasmic reticulum.
Could you tell the structural differences between these
two ribosomes in eukaryote? Can you predict it from thepresent situation?
Prof. Steitz: Actually, both prokaryotic ribosomes andeukaryotic ribosomes will secrete through the translocon,
so there are some similarities there for both eukaryotes
and prokaryotes. There are some differences as well, forsure. But the translocon itself is what is embedded in themembrane, and the ribosome sits on top, and so probably
the best structural work that is out there are the moment
is from Joachim Frank’s lab, published last year. At
about 12 Å resolution. They can actually fit two translocon molecules into the translocon density and they can
fit a ribosome onto it, and they can actually see the polypeptide going down the tunnel and going through thetranslocon. The problem is, again, detail. You cannot see
what the interactions are in detail. The translocon is interacting with the ribosome through a couple of RNA
loops that are coming down. So the ribosome itself stays
pretty clear of the membranes, certainly the non-polar
part of the membrane. So I do not think that the membrane itself is going to alter the ribosome structure. As
far as I know, there is no evidence so far from the EM

Keio J Med 2008; 57 (1): 1－ 14

that there is a significant difference in the ribosome
structure that results from the interaction with the translocon.
Chairman: Any other questions? Yes.
Unidentified speaker: How did you carry out the genetic study of the large ribosome; for example, how do you
generate mutations?
Prof. Steitz: That is a longish story. It gets particularly
long because the generation time of Haloarcula marismortui is extremely slow; it is half a day or so. Basically,
what they do is insert pieces of DNA selectively into thehaloarcula marismortui genome. Basically it is the same
approach to making mutations as is used in other systems. There are some complications having to do withthe fact that there are three copies of the 23S rRNA in
the chromosome. We were told when we started this
there were only two copies, but we discovered in factthat there were three. So the reason why, for example, on
one of the slides I said it was only 33% mutant 50S subunit is because it was easy or easier to replace one copy
initially, but we then got rid of a second gene copy andthat took a little bit more work. It is more or less standard genetics except that the haloarcula marismortui system itself is a little more of a challenge.
Chairman: Okay. One last question. Yes.
Mr.Ryo Miyakawa (An undergraduate student, Keio
University School of Medicine): You talked about theantibiotics designed to block the active site; however, I
thought you said the active site is very well conserved,
which means that in order to gain the resistance against
the antibiotics it takes only one or two mutations to get
the resistance.
Prof. Steitz: Yes.
Mr. Miyakawa: Why is there no antibiotics targeting the
not so well conserved areas of the ribosome? That way,
it will take a few more mutations, and therefore a lot
more time to gain resistance against the antibiotics.
Prof. Steitz: Those are both very good questions. I had
tried to indicate the answers, but I will be a little clearer
about it. The catalytic part of the peptidyl transfer centeris indeed very similar or essentially identical. Consequently some of those antibiotics, like blastocydin, areantibiotics in the sense that they will inhibit protein synthesis, they are just as effective against mice as they areagainst man as they are against bacteria, because thatpart is absolutely conserved, and indeed that is a bad target. In fact, it is not a target. Now, it turns out that a little
bit further down the tunnel in the region where I showed
the macrolides and some of those other antibiotics binding, and in the site where linezolid binds, there are somedifferences. The differences are sometimes one or two
nucleotides away from the surface, but they change theshape of the surface enough so that there is some specificity difference. Now as to the question of why not target some place that is completely different in the two

13

kingdoms: the reason they are structurally different isthat it does not matter so much to function what the nucleotide is there. So that means you can bind whatever
you want in that region, and it is not going to inhibit
function. Another binding site that is a great target if you
want to make a rat poison but is not so good if you wantto kill bacteria is the E site, because it turns out thestructure of the E site is very different in eubacteria andeukaryotes. There is a protein in eukaryotes that is in theregion and it is interacting with the CCA end of tRNA,and there are antibiotics―we are about to publish a paper showing several such antibiotics―that will bind
specifically to Haloarcula marismortui E site because ofcourse it looks like a eukaryote, but will not bind to eubacteria. That is nice except that you would actually liketo do it the other way around. It is not so clear to me thatthat is going to be easy to do, but it is a great place interms of difference. It is a big difference, and obviously
the E site is functionally important. So we are thinking
about that but it is not so obvious what to do.
Chairman: Okay, thank you very much, Tom.
Finally, I would like to make some words before closing this symposium. First of all, it is a really exciting andimpressive one-day symposium, and every speaker gavea very excellent talk. Then we learned lots about the
RNA from two different aspects. One aspect is about basic research of RNA science. The other additional aspect
is applied science, and particularly drug design and therapeutic development going well. So it seems to me thatnow we have an excellent high-tech car made of RNA,so this RNA car has an excellent two wheels. One wheelis the basic research wheel, and the other wheel is an applied science wheel. We have now lots of gasoline andwe can drive this wheel and rotate efficiently and coordinatedly. Hence this RNA car moves very elegantly andrapidly and is going forwards. So it seems this is a kindof stage of RNA science which we are doing in every research effort. This is something like kintaro ame in Japanese. Kintaro ame is a candy bar in which the same face
―Kintaro’s face, this is a superman―his face is printed
inside the bar. Therefore one slice at any position, there
appears the same face―Kintaro’s face. This is RNA.
Now this is it. Yesterday, Tom said that he had visited Japan more than 20 years ago, and at that time I also joined
the symposium seminar. It was Oji seminar held in Hokkaido, in Tomokomai. At that time, Joan was invited, andshe was working on splicing machinery, and she discovered a new small RNA which was important for the
splicing system, so she was really a big star at that moment. Tom accompanied Joan. Somehow I was really
impressed about you and we met for the first time there.
Next time, it was a very impressive occasion for me in1999; we all met in the small Greek island Spetses. Every three or four years, one advanced lecture course isorganized in the small island of Spetses. Marianne Grun-

14

Steitz TA : structures of the large ribosomal subunit

berg-Manago, my mentor, organized this lecture course.
On that occasion, Tom gave a five angstrom resolution
structure. The paper was published in Nature that year,
but really for me, it was the first time to listen to the angstrom atomic resolution. It was so impressive; it was oneof my most impressive lectures ever. So, many stories
and I am very happy to say that we shared some aspects
together in translational control fields. Finally, I would
like to express my big congratulations to Tom Steitz forreceiving the year 2006 Keio Medical Science Prize thisyear. I also would like to sincerely respect the committee
members in Keio University for their decision of thisprize, before Tom gets a Nobel prize, because I am very
confident that the Nobel prize will go to Tom Steitz.
Hence, this is a very, very excellent decision that Keio
University made. Finally, I would like to thank all the
speakers and audience for being here and stimulating this
discussion, and making it a very joyful meeting here, andfinally next door everyone is invited to the get-together
party with very nice food and very nice drinks ready andeverybody is invited to next door. So we would like toclose this symposium. Thank you very much.
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