Discovering Ribozymes
Transcript of Part 1: Discovering Ribozymes
00:00:30.07 Hi! I am Tom Cech. And I've been asked to talk about my favorite experiment. 00:00:34.27 Why did I pick this particular experiment? 00:00:37.04 Well... It was one that I did with my own hands, 00:00:41.06 and there is something special about discovering something through your own work in the lab. 00:00:46.03 That is, as much as I enjoy working with students and postdoctoral fellows 00:00:49.29 and exalt over their discoveries, 00:00:53.07 something special about being able to make a discovery by yourself. 00:00:58.00 This came when we were looking at initially the transcription 00:01:02.26 of a large ribosomal RNA precursor 00:01:07.02 in a single cell pond animal, Tetrahymena thermophila. 00:01:10.22 The gene that we were looking at made an RNA that 00:01:15.05 was interrupted by a stretch of non-coding sequence, 00:01:19.02 called an intron. And at this time, RNA splicing had been discovered a few years earlier, 00:01:26.02 but people knew very little about the mechanism by which an intron containing RNA 00:01:32.01 would be spliced to rejoin these two flanking sequences called exons 00:01:37.26 and release that intron RNA. Now we had found in our studies of transcription of this gene 00:01:44.09 that the RNA splicing was taking place outside of the cell, in vitro, with our purified extracted RNA. 00:01:54.08 So this seemed to be an opportunity for us to investigate the mechanism of RNA splicing. 00:02:03.05 Initially this splicing reaction was taking place in the same cocktail 00:02:09.14 of small molecules that were necessary for transcription. 00:02:13.27 So I decided that it would be useful to see which of those small molecules 00:02:20.08 was really required, not for the transcription part, but for the RNA splicing. 00:02:24.12 So I added them, I subtracted them one at a time from the transcription cocktail, 00:02:29.29 I added them back. And I found that there were only two small molecules that were required: 00:02:35.06 one was a simple salt, magnesium chloride, 00:02:39.01 and the other was one of the precursors of RNA itself, 00:02:44.06 guanosine triphosphate. 00:02:46.11 A lot of specificity here: G was required, A wouldn't do it, 00:02:51.23 C wouldn't do it, U wouldn't do it. It had to be guanosine. 00:02:56.08 And this seemed unusual, it seemed interesting, 00:03:01.17 but we didn't really know what to make of it 00:03:06.11 until someone else in the laboratory, Art Zaug, 00:03:08.27 was sequencing the end of the intron 00:03:12.29 after it was excised from the larger RNA. 00:03:16.13 And he found that the sequence of the cut out RNA 00:03:21.03 started with a guanosine residue which is one of the four nucleotides of RNA. 00:03:27.25 We didn't think much of it until we found out from Joe Gall's laboratory 00:03:32.00 at Yale university, that they had sequenced the gene that encodes this RNA, 00:03:38.14 and that they were adamant that there was no guanine 00:03:44.05 present near that exon-intron boundary. 00:03:47.26 The rest of our sequences agreed between the two laboratories, 00:03:53.19 and so here we had a situation where it looked like maybe 00:03:57.03 there was a guanine that wasn't encoded by the DNA 00:04:01.23 that was added to the end of the intron. 00:04:04.13 And at the same time we had this evidence 00:04:07.09 from my earlier work that G was a required ingredient in the splicing reaction. 00:04:13.07 So maybe these two observation were related to each other, 00:04:18.20 although the idea that one could just add a guanosine triphosphate 00:04:23.28 to purified RNA and expect any chemical reaction, 00:04:28.22 any cutting and joining reaction, to take place seemed unprecedented 00:04:34.20 and really quite farfetched. So, at the time 00:04:38.19 I did this reaction rather, this experiment, rather quietly. 00:04:42.14 I didn't tell any of the graduate students what I was doing. 00:04:46.20 I didn't want to look foolish if this reaction 00:04:49.09 failed as it was probably destined to do. 00:04:53.10 When I added in one tube radiolabeled GTP to the purified precursor RNA 00:05:01.19 and in other tubes the other nucleotides 00:05:04.27 finally in the midst of my teaching schedule I had an opportunity to run the gel 00:05:09.10 to look at the splicing products, and lo and behold, 00:05:12.08 only in the lane where I had added the radiolabeled GTP 00:05:17.03 was there a radiolabeled RNA band the exact size of the intervening sequence. 00:05:23.29 So, I ran back to my office to try to get a little bit of peace and quiet 00:05:31.07 to try to draw out what must be happening in terms of the splicing mechanism. 00:05:36.08 and what I quickly came up with was that this guanine that was added during the splicing reaction 00:05:44.17 was being joined to the 5' end of the intron. 00:05:48.24 So it looked like it was attacking the splice site phosphate, 00:05:53.24 and forming a new oxygen-phosphorus bond that hadn't been there before. 00:05:58.16 Now if that happened, that would explain how the GTP would be covalently bound to 00:06:04.27 the end of the intron. But what would happen with the other product of this reaction? 00:06:10.07 Well, the 5' exon would then have to be released with a hydroxyl group at its 3' end. 00:06:18.04 And exactly the same kind of chemical step if it occurred now 00:06:23.15 between this exon and the downstream splice site 00:06:26.22 would result in ligation of the exons and release of the intron RNA 00:06:33.24 with this diagnostic guanosine at its 5' end. 00:06:39.28 And, so, I thought, well, has anyone... is this even chemically reasonable? 00:06:45.26 So, fortunately I had an organic chemistry textbook within reach, 00:06:50.04 pulled it out. It did not discuss this sort of reaction 00:06:53.22 with phosphate esters, but with esters of carbon, this was simply a trans-esterification reaction. 00:07:03.03 So, what was happening...so there was precedent for this sort of reaction. 00:07:07.01 You start out with one ester linkage, 00:07:10.18 in this case a phospho-diester linkage, and you are now using 00:07:15.13 the hydroxyl group of the ribosugar of guanosine 00:07:19.27 as a nucleophile attack at this site. 00:07:24.14 It is also called an Sn2 reaction as you probably studied in your organic chemistry class. 00:07:31.16 And this swapping of partners in this ester linkage 00:07:36.12 turned out to be the key mechanism of RNA splicing. 00:07:41.04 So although it was exciting to have this mechanistic information 00:07:45.03 about RNA splicing, the question of the catalyst that was allowing all of this to happen eluded us 00:07:51.23 for another year. We were assuming that there had to be a protein enzyme 00:07:56.18 that was responsible for a reaction that took place with this incredible specificity, 00:08:02.07 after all in this long RNA there was only this one site that was being chosen as a splice site. 00:08:08.16 There was also specificity with respect to guanosine 00:08:11.02 relative to the other nucleotides. And the reaction was speeded up 00:08:16.20 many billions of fold faster than 00:08:20.08 a spontaneous phospho-transesterification reaction 00:08:22.22 would be predicted to occur. 00:08:25.14 So, if all biological catalysts are protein enzymes, where was the protein? 00:08:31.05 And we spent a lot of time looking for protein contaminants in our pure RNA preparation. 00:08:37.03 Finally, out of a lack of being able to identify any, we switched around the hypothesis 00:08:44.09 and said, maybe it is just the RNA that is folding up to form the catalytic center for this reaction. 00:08:51.02 To test that idea we were able to make an artificial transcript 00:08:55.25 that had never seen the inside of a Tetrahymena cell 00:08:59.01 and when we added guanosine triphosphate to that artificial RNA 00:09:03.21 the addition of G to the end of the intron 00:09:08.04 and the RNA splicing that ensued convinced us that it was time to announce 00:09:13.12 that RNA could be an enzyme, that RNA had catalytic activity. 00:09:19.15 This turned out to be the experiment, or the set of experiments, 00:09:23.23 that resulted in the Nobel Prize in Chemistry 8 years later, 00:09:28.24 but I think it is important to understand that at the time, 00:09:32.13 we were not driven by the possibility of getting awards 00:09:35.19 or recognition. It was simple curiosity about how does RNA splicing occur 00:09:43.12 and how could this reaction even be occurring with pure RNA 00:09:48.27 that was driving us in the laboratory 00:09:52.05 and giving us so much satisfaction.