Discovering Ribozymes

Top

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.

This Talk

Speaker: Tom Cech

Audience:

General Public
Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: October 2010

Educator Resources for this Talk

Talk Overview

In this talk about discovering ribozymes, Tom Cech describes the excitement of doing the experiments that led to the discovery that RNA can catalyze the cutting and rejoining of chemical bonds. Prior to this discovery, most scientists believed that only proteins catalyzed biological reactions. The importance of this work was recognized with the 1989 Nobel Prize in Chemistry.

Speaker Bio

Tom Cech

Tom Cech has been on the faculty of the University of Colarado, Boulder for more than 30 years. In 2000, he was named president of the Howard Hughes Medical Institute and remained in this position until 2009. Currently, his lab investigates the structure and replication of telomeric DNA. Continue Reading