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COMMEMORATIVE LECTURE Structural Insights into the Functions of the Large 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 Prof. Thomas A. Steitz, Department of Molecular Medical Science, University of Tokyo): Finally we will Biophysics and Biochemistry and Department of have a lecture by Prof. Thomas A. Steitz. He is the win- Chemistry, Yale University, and Howard Hughes ner of Keio Medical Science Prize. Already, he explained Medical Institute: It is a great pleasure to be here, and or talked about his personal history and therefore really I thank you so much for that kind introduction. As I said do not need to repeat the same thing; but briefly I would yesterday, it is always wonderful to come to Japan and like to introduce his career. He graduated in Chemistry visit. My son and I had a great breakfast this morning; a from Lawrence College,Wisconsin in 1962, and he fin- sushi breakfast at the fish dock,*1 the best sushi that I ished his PhD in biochemistry and molecular biology at ever had in my life; it is going to be hard to do this again, Harvard University. Then he started his professional ca- I have to say, back home. My son is out having a good reer path in Harvard University, the same department of time in Tokyo and I am here having to talk, so I am go- chemistry, and he also spent three years 1967-1970 in ing to have a good time too. Cambridge, England, where he met so many important I would like to start out by giving an introduction to the figures as we learned yesterday. Then he got an appoint- ribosome and the ribosome problem, and then provide a ment in Yale University in molecular biophysics and bio- few details about how we solved the structure of the chemistry in 1970. Since then he has stayed in the same large ribosomal subunit. Now, I realize that this is an un- department, and carried up through an assistant profes- specialized audience with respect to structure determina- sorship, associate professorship and full professorship. tion, with one or two exceptions obviously here, but I He has received quite many awards. Among those just I want to give a sense of what the problem was that needed will mention a few important points. In 1990, he became to be solved and how we went about it, but that will only a member of the National Academy of Sciences in the be five slides, so not to worry. Then I shall talk about United States, and also the same year a member of the what we have done to understand the mechanism of pep- American Academy of Arts and Sciences. He received tide bond formation. Finally, I shall talk about the antibi- several awards: for example, in the year 2001, the Rosen- otic binding studies that we have done and the examina- stein Award for Distinguished Work in Basic Medical tion of the effect of resistance mutations, and finally talk Science, and the latest one in the year 2004, the Frank about what Rib-X Pharmaceuticals, Inc.―the company Westheimer Medal of Harvard University. As you know, that I and some of my colleagues started five years ago this year he received the Keio Medical Science Prize. He ―are doing to use this information to design new antibi- received it; I trust and I hope that this award is the best otics, which looks like it is going to be successful. and prestigious award. Now we will ask Tom for this lec- I shall start with a fact published in Nature a couple of ture about “Structural insights into the functions of the years ago, that in the United States, two million people large ribosomal subunit, a major antibiotic target.” will acquire antibiotic resistance infections in US hospi- tals, 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 2 Steitz TA : structures of the large ribosomal subunit problem. Another important point is that the ribosome is another 1,500 in the small subunits; so the whole assem- a major target of antibiotics―in fact, the major target― bly of the 70S is about two and a half million molecular and the large ribosomal subunit is the major target in weight, two-thirds RNA by mass. Think of it as an RNA protein synthesis. Consequently, our understanding of machine contaminated with proteins, whereas the spli- how a large ribosomal subunit works and how it is inhib- ceosome, I tell my wife,*2 is a protein machine contami- ited by antibiotics should indeed to be helpful to attack- nated with RNA. The ribosomes constitute one-fourth of ing this problem. the bacterial cell mass, so it is very abundant, and of But let me back up and say a few things about our ap- course that is extremely useful if you want to do struc- proach. There are some differences of opinions about tural studies, to have a very abundant machine. how one should do structural biology. This is my opin- Now the early structural work in the mid-70’s was ion, which was exhibited very well by Dr. Yokoyama a done by Jim Lake using negative staining in electron mi- couple of lectures ago. That is: you need to have the croscopy, and he was able only to show the shapes of the structures of every stage of a process in order to under- two subunits, but his shapes for the individual subunits stand that process. It is not just good enough to have one are largely right at this resolution of 50 Å or so; then ex- structure; you need to have them all. So it certainly is not amining the model of the whole ribosome you can see enough to look at the pieces. As you know, most assem- that the two subunits are sort of snuggling up here like a blies are made from a lot of pieces. It is easy for us to be pair with the small subunit lying on top of the large sub- reductionists; we all reductionists, and we are tempted to unit. Using cryo-electron microscopy (cryo-EM), it was look at whatever we can get our hands on, but knowing possible to go to somewhat higher resolution by 1997 the structures of the pieces of a clock does not tell you and position these transfer RNA (tRNA) molecules ap- how a clock works. But I might say that even if you proximately correctly, showing the CCA going into the know the structure of a clock sitting there, that does not large subunit and the anticodon loops of decoding a fic- tell you how a clock works either. So what you have to tionally placed messenger RNA, and then the E-site do is have the structures of the clock or the assembly that tRNA, and more or less over here but again not exactly works at each step in that process. Even though some of positioned correctly. So that is where we were in our un- the steps are metastable, you have to be a clever bio- derstanding of ribosome structures approximately 10 chemist and figure out how to stabilize that state so you years ago. can take a snapshot of it, and then you can put it together I have met up with Venki Ramakrishnan many times― into a movie. So that is the philosophy that we have tak- actually, he was a post-doc at Yale in the 1980s with Don en with all the problems that we were studying in the lab. Engleman and Peter Moore―but I met him in Sweden at I did show some movies yesterday, and that is how we a meeting, so we had a photo opportunity, and I am go- are looking at the ribosome as well. ing to use Venki’s structure of the small ribosomal sub- We have been looking at all aspects of gene expression, unit and put it together with our structure of the larger ri- but of course today I am just going to focus on the ribo- bosomal subunit to get the structure of the whole 70S ri- some and indeed I am only going to focus on the large ri- bosome. The two subunits were positioned using the co- bosomal subunit where peptide bond formation occurs. ordinates of the phosphate backbone of the 70S from This schematic drawing of protein synthesis by Jim Wat- Harry Noller and his three A-site, P-site, E-site tRNAs. sonshows where our understanding of the ribosome was Venki, as you may have noticed if you are paying atten- in 1964. It was known that the decoding happens in the tion to this area, has recently determined at 2.8 ang- small subunit and peptide bond formation occurs on the stroms resolution the structure of the 70S assembly with large subunit. Also shown was the A site, where the ami- tRNAs bound in the P site and E site and partially the A noacyl-tRNA binds, and the P site, where the peptidyl- site (5); so there is an even more accurate structure of tRNA binds, which he proposed existed, but he did not this assembly than shown in Figure 1 but I think for our know about the E site, where the exiting deacylated present purpose you would not be able to tell the differ- tRNA binds, and he did not know about the polypeptide ence, nor would I.

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