the rna world hypothesis
DESCRIPTION
The RNA World Hypothesis. -cofactors -ancient enzymes -ribozymes. Ribozymes. “Catalytic RNA”. Nobel prize - Chemistry 1989. T.R. Cech (Colorado, HHMI). S. Altman (Yale). RNase P. Tetrahymena group I. http://almaz.com/nobel/chemistry/1989b.html. - PowerPoint PPT PresentationTRANSCRIPT
DNA
RNA
Proteins
The RNA World Hypothesis
activityinformation-cofactors-ancient enzymes-ribozymes
Ribozymes“Catalytic RNA”
Nobel prize - Chemistry 1989
T.R. Cech (Colorado, HHMI) S. Altman (Yale)
RNase PTetrahymena group I
http://almaz.com/nobel/chemistry/1989a.htmlhttp://almaz.com/nobel/chemistry/1989b.html
Ribozymes“Catalytic RNA”
-rate enhancement-turnover
Catalysis
Small:hammerheadhepatitis hairpinVS
Medium:group Igroup IIRNase P
Large:ribosomespliceosome?
Auto-catalysis
-rate enhancementon self reaction
Catalysis
A B+ C
A B+
AB‡
AB‡
C A B+
AB‡
D
C
Acceleration
CatalysisTurnover
A B+ Ccat cat
RNA Catalysis
How fast?
Ribozyme
Group IHammerheadRNA ligase
Enhancement(kcat/uncat)
Turnover(kcat) min-1
1011
106
109
0.1>1100
Enzyme
T7 RNA polDNA ligase
1011
10920,000
28
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?
Splicing
Exon ExonIntron
Tetrahymena intron removal
Two sequential transesterifications
UpA GpU
G-OH
GpA GpU
U-OH
GpA G-OH
UpU
5’ 3’
5’
3’
5’
3’5’
5’ splice site
Stereochemical Course of Splicing
-transesterification reactions of splicing are nucleophilic substitution reactions
Recall:
LGNu Nu LG
inversion
LGNu Nu LG
Nu Nu
RetentionInversion
mirror images
R S
OR
O
OR'
-O
OR
S O
OR'
-OR
S
OR'
-O
5’
3’ Rp Sp
Prochiral Centre Made Chiral by S Substitution
P P P
Stereochemistry of Group I Splicing
-Rp at 5’ splice site gives Sp in product
-Sp at 3’ splice site gives Rp in product
X
P OO(S) -
Y
GpZ
G-OH
X-OH
P
O-
O(S)
Y
GpZ
G
X-OH
P
O-
O(S)
ZG
GpY
X
P OO(S) -
Z
Rp
Sp Sp
Rp
Stereochemistry of Group I Splicing
Tetrahymena intron removal
UpA GpU
G-OH
GpA GpU
U-OH
GpA G-OH
UpU
5’ 3’
5’
3’
5’
3’5’
NuLG
One Active Site in Group I Intron
first step
second step
G Binding Site - Group I Intron
G Binding Site
G264
C311
N
NN
N
NN
H N
O
H
O
NH
H
H
N
NN
N
NN
HN
O
H
O
NH
H
HN
N
NN
H
O
N
H
H
GG
G
Specificity Swap at G Binding Site - Group I Intron
- mutate G-C to A-U
N
NN
N
H
O
NH
H
N
NN
N NH
H
H
H
NC
N
CH2
N
NH3C3H5CO2+ -
+H
H
H
H
OC
N
CH2
NH3C3H5CO2+ -
H
H
guanosine 2-aminopurine
arginine citrulline
G-C A-U
G co-factor
2AP co-factor
+ arginine
+ citrulline
+
+
+
+
--
-
-
N
NN
N
NN
H N
O
H
O
NH
H
H
N
N
NN
H
O
N
H
H
N
NN
N
NN
O
O
N
N
N
NN
N
H
H
H H
H
N
NN
N
H
O
NH
H
N
NN
N
NH H
H
H
NC
N
CH2
N
NH3C3H5CO2+ -
+H
H
H
H
OC
N
CH2
NH3C3H5CO2+ -
H
H
G
C
G
AP
A
U
A
G
Arg
Cit
Creation of a Ribozyme
G
#•••••
IGS
G
• • • • •IGS
309
265P3P8 P7
274277
99
5'
3'
P4
[P2]
P5P6b P6a
P9
P5b P5a
P5c
J8/7301
108 112
327
330
252 254
312305
267
95
Secondary Structure of Group I RNA
P4-P6 X-ray
Science (1996) 273: 1678-85
Group I X-ray
Science (1998) 282: 259-64
Group I Intron X-ray
Nature 430, 45-50 (1 July 2004)
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Group I Intron X-ray
Nature 430, 45-50 (1 July 2004)
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Group I Intron X-ray
Nature 430, 45-50 (1 July 2004)
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Satellite Tobacco Ringspot Virus RNA
C
NNN
AAA
G
NNNN
AG
NN
CUG
A
NNN
NNN NN• • • • •
• • •
GU
•• N
O
OHO
P
O
O
O-
N
O
OO
N
P
O O-
.
C
NNN
AAA
G
NNNN
AG
NN
CUG
A
NNN
NNN NN• • • • •
• • •
GU
•• N
#
Design of a Ribozyme
C
NNN
AAA
G
NNNN
AG
NN
CUG
A
NNN
NNN NN• • • • •
• • •
GU
•• N
5'3'
3'5'
ribozyme
substrate
Hammerhead Ribozyme
UG
CU
C
CC
GC
GC
GG
UC
UGA
GUCCG
GAC
ACA
GG
AA
5'3'
XA
5' 3'
3'5'
O
OHO
P
O
O
O-
N
O
OO
N
P
O O-
Hammerhead X-ray
Cell (1995) 81: 991-1002
UG16.2
CU
C
CC
GC
GC
GG
UC
UG5A
GUCCG
GAC
ACA
GG
AA
5'3'
XA
5' 3'
3'5'
G5
G16.2
(a) (b)
N
NN
N
H
O
NH
H
N
NN
N
H
O
H
guanosine inosine
Chemistry of Hammerhead Cleavage
Cell (1998) 92: 665-73
X
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Altered Hammerhead Conformation
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Molecular Cell, Volume 23,4, 447-450,2006
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Altered Hammerhead Conformation
Cell, 126, 2, 309-320, 2006
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Altered Conformation: Active Site
Hepatitis d Ribozyme
Hepatitis d X-ray
Nature (1998) 395:567-74
Hepatitis d X-ray
Nature (1998) 395:567-74
General Base Catalysis in a Ribozyme
Science (1999) 286: 123-6
Imidazole Rescue of Ribozyme Activity
Science (1999) 286: 123-6
Imidazole Rescue of Ribozyme Activity
Science (1999) 286: 123-6