The Role of RNA as a Catalyst

1. The Role of RNA as a Catalyst Zhenzi Lee 12/11/2005

2. The role of nucleic acids and proteins • In 1970s: Proteins made things happen in the cell Nucleic acids informational molecules Enzymes accelerated the rates of chemical reactions

3. Then,in 1981,a paper was published that blur the distinction

4. Cech’s work • Thomas Cech and his co-workers at the University of Colorado had been studying the process of pre-RNA spilcing of the ciliated protozoanTetrabymena thermophila ,they found the isolated nuclei can carry out the entire splicing reaction.

5. Pre-knowledge… • RNA Splicing  Gene Splicing of an intron is a complicated reaction that requires recognition of the sequences that border the intron,cleavage of the phosphodiester bonds on both sides of the intron,and ligation of the adjoining fragments. Intron1 Intron2 Exon 1 Exon 2 Exon3  Primary transcribe Pre-RNA Intron1 Intron2 Exon 1 Exon 2 Exon3  Mature transcribe Exon 1 Exon 2 Exon3

6. The steps of Cech’s work Isolate pre-RNA Incubated in a medium with some nuclear components (without protein) When there were high concentration of NH4+ in the presence of Mg2+ and a guanosine phosphate(e.g. ,GTP)… The intron was spliced! A protein was bound very tightly to the RNA, or the pre-rRNA molecule can splicing itself?

7. An artificial system to resolve the question Transform the DNA that encodes the rRNA precursor to E.coli Purify the DNA Use as a template for in vitro transcription by RNA polymerase The pre-rRNA synthesized in vitro is then purified and incubated with ions and a guanosine compound The splicing reaction is done！

8. The “ribozyme” • As a result,RNA was shown to be capable of catalyzing a complex,multistep reaction. • In the case of Cech,the RNA acted on itself rather than on an independeant substrate. • Cech called the RNA a “ribozyme”

9. Another discovery • Later,a second example of RNA catalysis was discovered by Sidney Altman of Yale University and Norman Pace of the National Jewish Hosptial in Denver • They found ribonuclease P, an enzyme involved in the processing of a transfer RNA precursor in bacteria, was also an “ribozyme”, and it acted on another molecule as most enzymes do. Picture: see page 486

10. The old question Which component of the large ribosomal subunit is the peptidyl transferase, that is,the catalyst for peptide bond formation?

11. Noller’s work Purified the ribosomes from Thermus aquaticus Treated preparations of the large ribosomal subunit with SDS,proteinase K and several rounds of phenol to remove 95% of the protein The RNA can retained 80% of the peptidyl transferase activity It is generally agreed that these experiments demonstrated that peptidyl transferase is a ribozyme

12. Later demonstration Treat large ribosomal subunit with CCdA-phosphate-puromycin which can inhibits peptide bond formation by binding to the peptidyl transferase active site Determined the location of the bound inhibitor at atomic resolution It was found within a cleft of the subunit that is surrounded entirely by conserved nucleotide residues of the 23S rRNA Ribosome is a ribozyme Picture:see page 436

13. Thank you for your attention!