Triplex Structures in an RNA Pseudoknot
Enhance Mechanical Stability and Increase
Efficiency of –1 Ribosomal Frameshifting
Gang CHEN
Department of Molecular Biology
The Scripps Research Institute
5 October, 2009
One amino acid in a protein is coded by three
nucleotides in a messenger RNA
0 frame:
A U G
Start codon
U A G
Stop codon
Ribosomal reading frames
+1 frame: A U G
U A G
0 frame:
A U G
U A G
–1 frame: A U G
U A G
×10–5
Intrinsic frameshift error: <5×
Structures in mRNA coding regions have
to be unfolded for translation
Loop 2
Stem 2
5'
3'
Stem 2
5'
3'
Hairpin
5'
3'
Stem 1
Loop 1
Pseudoknot
(2D)
Puglisi, Wyatt, and Tinoco, (1988) Nature
Pseudoknot
(3D)
Structure of prokaryotic ribosome
Korostelev, Ermolenko, and Noller (2008) Curr. Opin. Chem. Biol.
Three components for programmed –1 ribosomal
frameshifting
slippery
sequence
0 frame:
single strand spacer
(5-10 nucleotides)
X XXY YYZ
start codon
–1 frame:
XXX YYY Z
start codon
X is any nucleotide, Y is A or U, and Z is usually not G
pseudoknot
Pseudoknot structure stimulates –1 ribosomal
frameshifting
Drawing is not to scale
Programmed –1 ribosomal frameshifting is
utilized by many viruses
•
Human immunodeficiency virus (HIV) (–1 frameshifting
efficiency: <10%).
•
Severe Acute Respiratory Syndrome coronavirus (SARSCov) (–1 frameshifting efficiency: ~15%).
•
Novel anti-virus therapy may be developed by targeting –1
frameshifting.
Choosing a model pseudoknot to study
–1 ribosomal frameshifting
•
High resolution three dimensional structure
•
High efficiency (>40%) in stimulating –1 frameshifting
NMR structure of human telomerase RNA
pseudoknot (∆U177)
Theimer, Blois, and Feigon, (2005) Mol. Cell
Base triples in human telomerase RNA
pseudoknot (∆U177)
Minor groove
base triples
Major groove
base triples
Theimer, Blois, and Feigon, (2005) Mol. Cell
Major groove base triples
U.A-U
C+.G-C
Human telomerase RNA pseudoknot (∆U177) as
a model system to study –1 frameshifting
•
Are pseudoknot base triples required for high efficiency
–1 ribosomal frameshifting?
•
How do base triples affect mechanical stability of RNA
pseudoknot?
The construct for bulk frameshifting assay
start codon
In vitro translation in rabbit
reticulocyte lysate (Promega)
0 frame stop codon
The construct for bulk frameshifting assay
start codon
0 frame:
Smaller protein
–1 frame:
Larger fusion protein
In vitro translation in rabbit
reticulocyte lysate (Promega)
0 frame stop codon
–1 frame stop codon
Pseudoknot mutants based on ∆U177:
Mutations in loops
Pseudoknot
G
U
C
C
C
Number
of base
triples
disrupted
∆U177
0
TeloWT (with U177)
0
100C115C174G
0
101C114C175G
0
115C174G
1
114C175G
1
101C
1
102C
1
GU
2
CCC
3
CCCGU
5
Pseudoknot mutants based on ∆U177:
Mutations in loops
Pseudoknot
101C
102C
Number
of base
triples
disrupted
∆U177
0
TeloWT (with U177)
0
100C115C174G
0
101C114C175G
0
115C174G
1
114C175G
1
101C
1
102C
1
GU
2
CCC
3
CCCGU
5
Pseudoknot mutants based on ∆U177:
Mutations in stem 2
Pseudoknot
115C174G
114C175G
Number
of base
triples
disrupted
∆U177
0
TeloWT (with U177)
0
100C115C174G
0
101C114C175G
0
115C174G
1
114C175G
1
101C
1
102C
1
GU
2
CCC
3
CCCGU
5
Pseudoknot mutants based on ∆U177:
Mutations in stem 2 and loop 1
Pseudoknot
100C115C174G
101C114C175G
Number
of base
triples
disrupted
∆U177
0
TeloWT (with U177)
0
100C115C174G
0
101C114C175G
0
115C174G
1
114C175G
1
101C
1
102C
1
GU
2
CCC
3
CCCGU
5
Pseudoknot mutants based on ∆U177:
Mutations in stem 2
Pseudoknot
U177 bulge
Number
of base
triples
disrupted
∆U177
0
TeloWT (with U177)
0
100C115C174G
0
101C114C175G
0
115C174G
1
114C175G
1
101C
1
102C
1
GU
2
CCC
3
CCCGU
5
∆U177
101C114C175G
100C115C174G
114C175G
102C
115C174G
CCCGU
CCC
GU
Frameshifting efficiency measured by
SDS-PAGE
5 3 2 1 1 1 0 0 0
Number of base triples disrupted
53
50
45
23
33
16
3
2
0.1
–1 frame product
0 frame product
–1 frameshifting efficiency (%)
Pseudoknot base triples are required
for high-efficiency –1 frameshifting
Pseudoknot base triples are required for
high efficiency –1 frameshifting
C
C
C
101C
102C
100C115C174G
115C174G
114C175G
101C114C175G
U177 bulge
0
1
>1
60
50
40
30
20
10
0
10 D
1C elta
10 1 U
0C 14 17
C 7
11 ∆U177
1
5C 75
17 G
101C114C175G
Te 4
lo G
100C115C174G
W
11TeloWT
10 T
4 2
11 C102C
17 C
5C 5
17 G
114C175G
4G
1
115C174G
01
C
101C
G
U
CGU
C C
CCCC
C
C
G
U
CCCGU
G
U
-1 frameshift efficiency (%)
# of base triples
disrupted:
Pseudoknot construct
Bulk studies:
•
Pseudoknot base triples are required for high
efficiency (>40%) –1 ribosomal frameshifting.
Single-molecule studies:
•
How does RNA pseudoknot unfold and fold?
•
How do base triples affect mechanical stability of
pseudoknot?
•
Is there correlation between pseudoknot mechanical
stability and –1 ribosomal frameshifting efficiency?
Bulk thermal unfolding pathway
Stem 1 hairpin
Folding intermediate
Native pseudoknot
High temperature
Medium temperature
Low temperature
Theimer, Blois, and Feigon, (2005) Mol. Cell
Optical trap behaves as a linear Hooke spring
F = –k∆X
1 kcal/mol corresponds to ~7 pN•nm or 1.7 kBT (298 K)
Moffitt, Chemla, Smith, and Bustamante, (2008) Annu. Rev. Biochem.
Single-molecule mechanical (un)folding of RNA
•
Mechanical force is a unique physical variable which is
applied locally to RNA structures.
•
RNA folding and unfolding can be studied in near
physiological solution and at room temperature.
•
Kinetic intermediates can be directly revealed.
Optical tweezers for single-molecule
manipulation of RNA pseudoknots
Force
Force
Streptavidin-coated
pipette bead
5′
5′
592-bp RNA/DNA handle
RNA pseudoknot
543-bp RNA/DNA handle
3′
3′
Force
Anti-digoxigenin-coated
trap bead
Force
200 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.3, 22 ºC
Three classes of unfolding trajectories
50
Extension increase
Unfolding force
Force (pN)
40
30
a
20
b
c
d
e
f
g
Hop
10
40 nm
0
Extension (nm)
i
∆U177
∆U177
50
40
High-force 1-step
unfolding
30
The second step of
2-step unfolding
20
Low-force 1-step
unfolding
10
6
8
10
12
14
16
18
Extension change (nm)
20
Pulling rate: 100 nm/s
22
Unfolding force (pN)
Unfolding force (pN)
h
60
60
Mutant CCC
50
High-force 1-step
unfolding
40
30
The second step of
2-step unfolding
20
Low-force 1-step
unfolding
10
6
8
10
12
14
16
18
Extension change (nm)
m
20
22
Three classes of unfolding trajectories
CCC mutant
50
Extension increase
Unfolding force
Force (pN)
40
30
a
20
b
c
d
e
f
g
Hop
10
40 nm
0
Extension (nm)
60
i
∆U177
∆U177
50
40
High-force 1-step
unfolding
30
The second step of
2-step unfolding
20
Low-force 1-step
unfolding
10
6
8
10
12
14
16
18
Extension change (nm)
20
Pulling rate: 100 nm/s
22
Unfolding force (pN)
h
Unfolding force (pN)
C
C
C
60
Mutantmutant
CCC
CCC
50
High-force 1-step
unfolding
40
30
The second step of
2-step unfolding
20
Low-force 1-step
unfolding
10
6
8
10
12
14
16
18
Extension change (nm)
m
20
22
NMR structure of stem 1 hairpin
Theimer, Finger, Trantirek, and Feigon, (2003) Proc. Natl. Acad. Sci. U.S.A.
Isolated stem 1 and stem 2 hairpins for singlemolecule studies
3'
5'
3'
5'
Isolated stem 1 hairpin (29 nt)
Isolated stem 2 hairpin (30 nt)
Analysis of the second step of two-step
unfolding forces
60
Delta U177
Low force two-step
reaction (2nd step)
Unfolding force (pN)
∆U177
High force one-step
reaction
50
40
30
Low force one-step
reaction
20
10
6
8
10
12
14
16
18
20
22
Extension change (nm)
Which stem is disrupted during the second step of
low force two-step unfolding?
Unfolding force comparison
50
30
∆U177
Stem 1 HP Stem 2 HP
25
Count
40
30
20
17.5 ± 1.0 pN
0
100
Count
Count
20
15
80
60
40
20
Isolated stem 1
hairpin
unfolding
16.8 ± 1.4 pN
0
10
40
5
10 nm
0
Extension (nm)
Count
Force (pN)
10
The second
step of ∆U177
unfolding
14.2 ± 1.2 pN
Isolated stem 2
hairpin
unfolding
30
20
10
0
8
10
12
14
16
18
20
Unfolding force (pN)
22
Unfolding force (pN)
Stem 1 is disrupted during the second step of low force
two-step unfolding
24
Single-molecule mechanical
(un)folding pathway
hairpin
aStem
Stem 11hairpin
b
?
pseudoknot
cNative
Native pseudoknot
Folding
(3 pN)
Unfolding
(15-20 pN)
Folding
(<10 pN)
Hopping
(10-20 pN)
Folding intermediate
Single
strand
Unfolding
(15-20 pN)
Unfolding
(50 pN)
Analysis of one-step unfolding forces to map
structural features of the folding intermediate state
60
Delta U177
Low force two-step
reaction (2nd step)
Unfolding force (pN)
∆U177
High force one-step
reaction
50
40
30
Low force one-step
reaction
20
10
6
8
10
12
14
16
18
20
22
Extension change (nm)
Are base triples and stem 2 formed at the folding intermediate state?
Base triples only affect mechanical stability of
native pseudoknots
60
50
40
30
20
10
0
30
CCCGU
19.4 ± 1.2
22.2 ± 5.4
CCC
G
U
20
19.6 ± 1.0
31.0 ± 3.0
0
8
GU
6
4
18.7 ± 1.1
33.9 ± 3.0
2
0
4
Count
C
C
C
Count
10
∆U177
3
2 17.8 ± 1.6
49.8 ± 1.9
1
0
0
5 10 15 20 25 30 35 40 45 50 55 60
Unfolding force (pN)
Unfolding force (pN)
Position of base triple matters
8
101C
6
17.9 ± 1.7
4
38.1 ± 2.8
2
102C
10
18.0 ± 2.2
45.4 ± 3.0
5
0
4
Count
101C
102C
Count
0
15
∆U177
3
2 17.8 ± 1.6
49.8 ± 1.9
1
0
0
5 10 15 20 25 30 35 40 45 50 55 60
Unfolding force (pN)
Unfolding force (pN)
At the intermediate state, base triples are
NOT formed and stem 2 is partially formed
Stem
1 hairpin
a Stem
1 hairpin
Folding
intermediate c
Native pseudoknot
b
Folding intermediate
Folding
(3 pN)
Unfolding
(15-20 pN)
Folding
(<10 pN)
Hopping
(10-20 pN)
Native pseudoknot
Single
strand
Unfolding
(15-20 pN)
Unfolding
(50 pN)
Force (pN)
Extension (nm)
Hopping of stem 1 hairpin and Folding
intermediate of ∆U177 observed at constant force
16
15
14
13
12
11
10
9
5 nm
H–S
0
1
2
3
0
1
2
3
Single Strand
4
4
Time (sec)
5
6
7
8
5
6
7
8
Stem 1 Hairpin
Folding Intermediate
Force (pN)
Extension (nm)
Hopping of stem 1 hairpin and Folding
intermediate of ∆U177 observed at constant force
16
15
14
13
12
11
10
9
5 nm
H – (I) – P
0
1
2
3
0
1
2
3
Folding Intermediate
4
4
Time (sec)
5
6
7
8
5
6
7
8
Stem 1 Hairpin
Native Pseudoknot
Similar mechanical stability observed for
UAU, C+GC, and UGC base triples
15
114C175G
10
18.8 ± 1.1
49.0 ± 3.1
5
U.G-C
114C175G
Count
0
30
101C114C175G
20
10
19.9 ± 0.8
C+.G-C
49.8 ± 3.2
0
101C114C175G
Count
4
∆U177
3
2 17.8 ± 1.6
49.8 ± 1.9
1
0
0
5 10 15 20 25 30 35 40 45 50 55 60
Unfolding force (pN)
Unfolding force (pN)
U.A-U
Structures of major groove base triples
U.A-U
C+.G-C
U.G-C
New folding intermediate structures appear
for pseudoknots with mutations in stem 2
15
114C175G
10
18.8 ± 1.1
49.0 ± 3.1
5
U.G-C
114C175G
Count
0
30
101C114C175G
20
10
19.9 ± 0.8
C+.G-C
49.8 ± 3.2
0
101C114C175G
Count
4
∆U177
3
2 17.8 ± 1.6
49.8 ± 1.9
1
0
0
5 10 15 20 25 30 35 40 45 50 55 60
Unfolding force (pN)
Unfolding force (pN)
U.A-U
At the new intermediate state, base triples
are NOT formed and stem 2 is fully formed
3 pN
Folding intermediate
3 pN
New folding intermediate
Native pseudoknot
Multiple folding intermediate structures
appear for TeloWT
20
TeloWT
15
10
45.0 ± 4.3
U177 bulge
Count
Count
5
0
4
∆U177
3
2 17.8 ± 1.6
49.8 ± 1.9
1
0
0
5 10 15 20 25 30 35 40 45 50 55 60
Unfolding force (pN)
Unfolding force (pN)
NMR reveals similar structures but different
dynamics for TeloWT and ∆U177
TeloWT
∆U177
Kim, Zhang, Zhou, Theimer, Peterson, and Feigon, (2008) J. Mol. Biol.
•
Triplex structures significantly enhance mechanical
stability of native pseudoknots.
•
Triplex structures do not form in the folding
intermediate states.
•
Is there correlation between native pseudoknot
mechanical stability and –1 ribosomal frameshifting
efficiency?
Correlation between native pseudoknot
mechanical stability and frameshifting efficiency
-1 frameshift efficiency (%)
70
60
∆U177
101C114C175G
100C115C174G
TeloWT (with U177)
50
40
102C
30
114C175G
20
10
CCCGU
0
15
20
115C174G
101C
CCC
GU
25 30 35 40 45 50
Average unfolding force (pN)
y = (0.19 ± 0.27)exp((0.11 ± 0.03)x)
R2 = 0.84
55
x = 0 pN,
y = 0.2 %
x = 57 pN, y = 100 %
Unwinding of mRNA pseudoknot by ribosome:
Rotation is needed
5′
3′
3′
5′
Frameshifting is enhanced by
stabilizing both stem 1 and stem 2
5′
3′
Conclusions
•
RNA pseudoknot unfolding and folding pathways were
revealed by single-molecule and mutational studies.
•
Both major groove and minor groove base triples of a
pseudoknot contribute to mechanical stability and –1
frameshifting.
•
Mechanical unfolding may mimic helicase activity of
ribosome on mRNA.
Acknowledgments
University of California, Berkeley:
Prof. Nacho Tinoco
Prof. Carlos Bustamante
Dr. Steve Smith
Dr. Jin-Der Wen
National Chung-Hsing University:
Prof. Kung-Yao Chang
Mr. Ming-Yuan Chou
The Scripps Research Institute:
Prof. David Millar
Prof. James Williamson
Prof. Larry Gerace
University of Rochester:
Prof. Douglas Turner
Prof. Tom Krugh
Prof. Scott Kennedy