Journal Article: T. R. Cech, et al. (Dec. 1981). "In Vitro Splicing of the Ribosomal RNA Precursor of Tetrahymena: Involvement of a Guanosine Nucleotide in the Excision of the Intervening Sequence." Cell, Vol. 27:487-496.
I would like to discuss here the winning of the 1989 Nobel Prize in Chemistry by T.R. Cech and S. Altman for their separate discoveries of RNA's catalytic properties. This post will be based on information that I used previously in a presentation, "Ribozymes Revisited: The Discovery of RNA Catalysis" for the Journal Club for Undergraduates in Biological Engineering and the Sciences (JCUBES) at Washington University in St. Louis on January 30, 2012. You can see more about that presentation here: http://wustljcubes.wordpress.com/2013/01/28/jcubes-presents-gregory-harrison/
Basically, I think that the experiments that led up to the winning of this 1989 Nobel prize were absolutely beautiful and the scientists who performed them, I believe, deserve more credit from us in the modern day for their fantastic deduction skills.
The first installment of "Ribozymes Revisited" will discuss the experimental design and discovery made by T.R. Cech in 1981. I think the single journal article that best represents the experiments performed by Cech at this time is his 1981 Cell publication, "In Vitro Splicing of the Ribosomal RNA Precursor of Tetrahymena: Involvement of a Guanosine Nucleotide in the Excision of the Intervening Sequence." To the right here, we have an image of the super cool model organism Tetrahymena thermophila. This protist is the organism that Cech was working on at the time of his discovery.
Cech was attempting to isolate the ribosomal RNA precursor of the 26S rRNA subunit. He knew several things about this 26S rRNA: there was a precursor molecule, and this precursor contained an intervening sequence (intron) which was spliced out to produce the final product. However, he found that it was pretty difficult to isolate the precursor molecule, he could only isolate the final product. He eventually succeeded in isolating the precursor molecule by subjecting the samples to certain conditions. His plan was to add the precursor rRNA to different fractions of a nuclear extract in order to identify and characterize a theoretical "splicing enzyme" which would carry out this rRNA maturation process. In order to purify the precursor, he subjected the sample to SDS-phenol extraction, sedimentation in a formamide-sucrose gradient, and protease treatment; these steps ensured that the final preparation contained only precursor rRNA with no associated proteins or other molecules.
Before Cech and his team could even test different nuclear extractions for the presence of an RNA splicing enzyme, the rRNA precursor "degraded" in the control environment, yielding the final product and the intervening sequence (visualized by gel electrophoresis). He was extremely surprised by this result, particularly considering the protein-denaturing steps that were taken to purify the rRNA. He proposed two explanations: first, there might be a splicing enzyme very closely associated with the RNA molecule, or second, that this experiment might represent a "novel case of an RNA-mediated reaction that requires no protein."
This suggestion, at the time would have been a real jaw-dropper. He even adds quickly after this statement a parenthetical, "(See Discussion)," as if he were afraid people would be so shocked by this claim that they would dismiss his work (a definitely possibility given scientists' knowledge of RNA at the time). Anyway, the actual paper is a joy to read, and is full of these little apologetic adjustments that really bring to light the revolutionary nature of this discovery. You can read it here. (Hopefully you have access to this database, sorry that I can't post the actual PDF...).
Stay tuned to iRNAbooks this weekend for Part 2 of "Ribozymes Revisited," discussing the equally beautiful and revolutionary story of S. Altman!!!
**Tetrahymena image from http://en.wikipedia.org/wiki/File:Tetrahymena_thermophila.png
Basically, I think that the experiments that led up to the winning of this 1989 Nobel prize were absolutely beautiful and the scientists who performed them, I believe, deserve more credit from us in the modern day for their fantastic deduction skills.
The first installment of "Ribozymes Revisited" will discuss the experimental design and discovery made by T.R. Cech in 1981. I think the single journal article that best represents the experiments performed by Cech at this time is his 1981 Cell publication, "In Vitro Splicing of the Ribosomal RNA Precursor of Tetrahymena: Involvement of a Guanosine Nucleotide in the Excision of the Intervening Sequence." To the right here, we have an image of the super cool model organism Tetrahymena thermophila. This protist is the organism that Cech was working on at the time of his discovery.
Cech was attempting to isolate the ribosomal RNA precursor of the 26S rRNA subunit. He knew several things about this 26S rRNA: there was a precursor molecule, and this precursor contained an intervening sequence (intron) which was spliced out to produce the final product. However, he found that it was pretty difficult to isolate the precursor molecule, he could only isolate the final product. He eventually succeeded in isolating the precursor molecule by subjecting the samples to certain conditions. His plan was to add the precursor rRNA to different fractions of a nuclear extract in order to identify and characterize a theoretical "splicing enzyme" which would carry out this rRNA maturation process. In order to purify the precursor, he subjected the sample to SDS-phenol extraction, sedimentation in a formamide-sucrose gradient, and protease treatment; these steps ensured that the final preparation contained only precursor rRNA with no associated proteins or other molecules.
Before Cech and his team could even test different nuclear extractions for the presence of an RNA splicing enzyme, the rRNA precursor "degraded" in the control environment, yielding the final product and the intervening sequence (visualized by gel electrophoresis). He was extremely surprised by this result, particularly considering the protein-denaturing steps that were taken to purify the rRNA. He proposed two explanations: first, there might be a splicing enzyme very closely associated with the RNA molecule, or second, that this experiment might represent a "novel case of an RNA-mediated reaction that requires no protein."
This suggestion, at the time would have been a real jaw-dropper. He even adds quickly after this statement a parenthetical, "(See Discussion)," as if he were afraid people would be so shocked by this claim that they would dismiss his work (a definitely possibility given scientists' knowledge of RNA at the time). Anyway, the actual paper is a joy to read, and is full of these little apologetic adjustments that really bring to light the revolutionary nature of this discovery. You can read it here. (Hopefully you have access to this database, sorry that I can't post the actual PDF...).
Stay tuned to iRNAbooks this weekend for Part 2 of "Ribozymes Revisited," discussing the equally beautiful and revolutionary story of S. Altman!!!
**Tetrahymena image from http://en.wikipedia.org/wiki/File:Tetrahymena_thermophila.png