2. The RNA World
Hypothesis
-cofactors
-ancient enzymes
-ribozymes

3. Ribozymes “Catalytic RNA” Nobel prize - Chemistry 1989
T.R. Cech (Colorado, HHMI)
S. Altman (Yale)
RNase P
Tetrahymena group I

4. Ribozymes “Catalytic RNA” Catalysis Auto-catalysis -rate enhancement
-turnover
-rate enhancement
on self reaction
Small:
hammerhead
hepatitis d
hairpin VS
Medium:
group I
group II
RNase P
Large:
ribosome
spliceosome?

5. Catalysis
Acceleration

6. Catalysis
Turnover

7. RNA Catalysis
How fast?

8. pre-rRNA processing
-In eukaryotes, the gene is not continuous..."functional" sequences are interrupted
"non-functional" sequences.
-First discovered for adenovirus pre-mRNAs (PA Sharp 1977)
-Intron/Exon structure (W. Gilbert) of eukaryotic genome
Tetrahymena rRNA contains one intron (IVS). How is it removed?
Exon
Exon
Intron

9. Tetrahymena intron removal
Two sequential transesterifications
5’ splice site 3’ 5’ 3’ 5’ 5’ 5’ 3’

10. Stereochemical Course of Splicing
-transesterification reactions of splicing are
nucleophilic substitution reactions
Recall:
inversion

11. mirror images R S
Inversion
Retention

12. Prochiral Centre Made Chiral by S Substitution5’ P P P 3’ Rp Sp

13. Stereochemistry of Group I Splicing
-Rp at 5’ splice site gives Sp in product
-Sp at 3’ splice site gives Rp in product

14. Stereochemistry of Group I SplicingRp Rp Sp Sp

15. Tetrahymena intron removal3’ 5’ Nu LG 3’ 5’ 5’ 5’ 3’

16. One Active Site in Group I Intron
first step
second step

17. G Binding Site - Group I Intron
C311 G G
G264 G

18. Specificity Swap at G Binding Site - Group I Intron
- mutate G-C to A-U
guanosine
2-aminopurine
arginine
citrulline

19. Arg G C G G AP
Cit U A A

20. Creation of a Ribozyme

21. Secondary Structure of Group I RNA

22. P4-P6 X-ray
Science (1996) 273:

23. Group I X-ray
Science (1998) 282:

24. Group I Intron X-ray
Nature 430, (1 July 2004)

25. Group I Intron X-ray
Nature 430, (1 July 2004)

26. Group I Intron X-ray
Nature 430, (1 July 2004)

27. Satellite Tobacco Ringspot Virus RNA

28. Design of a Ribozyme
substrate
ribozyme

29. Hammerhead Ribozyme

30. Hammerhead X-ray
Cell (1995) 81:

31. guanosine
inosine

32. Chemistry of Hammerhead CleavageX
Cell (1998) 92:

33. Altered Hammerhead Conformation

34. Altered Hammerhead Conformation
Molecular Cell, Volume 23,4, ,2006

35. Altered Conformation: Active Site
Cell, 126, 2, , 2006

36. Hepatitis d Ribozyme

37. Hepatitis d X-ray
Nature (1998) 395:567-74

38. Hepatitis d X-ray
Nature (1998) 395:567-74

39. General Base Catalysis in a Ribozyme
Science (1999) 286:

40. Imidazole Rescue of Ribozyme Activity
Science (1999) 286:

41. Imidazole Rescue of Ribozyme Activity
Science (1999) 286: