How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert...
Transcript of How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert...
![Page 1: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/1.jpg)
How RNA tells right from wrong:base pairs, tertiary interactions
and counterions
Sarah WoodsonJohns Hopkins University
![Page 2: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/2.jpg)
folding
10-100 ms
transcription
25 nt/s
hairpins10-100 !s
![Page 3: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/3.jpg)
folding
10-100 ms
misfolded
intermediate
slow
![Page 4: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/4.jpg)
Cations trigger rapid 3D collapse of RNA
Woodson, Curr. Opin. Chem. Biol. (2005)
![Page 5: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/5.jpg)
Kinetic partitioning: individualmolecules take different paths to N
fast
slow
U
I
N
U273A ! 80%
wt ! 98%
!
misfolded(inactive)
folded(active)
Thirumalai & Woodson, Acc. Chem. Res. (1996)
high tempurea
fast
slow
![Page 6: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/6.jpg)
Ribozyme core misfolds
Guo et al., Mol Cell (2004) Sclavi et al, Science; Pan et al., JMB (1998)
![Page 7: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/7.jpg)
Helix stability influences folding kinetics
Pan et al., J. Mol. Biol. 280: 597-609 (1998); J. Mol. Biol. 296: 133-144 (2000)
80% foldsrapidly (~1 s-1)
![Page 8: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/8.jpg)
N
I
U
G
‡
Native tertiary interactions stabilizemisfolded intermediates
Treiber & Williamson, J Mol Biol 305: 11-21 (2001); Treiber et al., Science279: 1943-1946 (1998); Pan & Woodson, J Mol Biol 294: 955-965 (1999)
P5abc
loop-loopinteractions
![Page 9: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/9.jpg)
Circular permutation of ribozyme
P2/P2.1
P4-P6P3-P9
P3*
P9.1/P9.250
100
150
200
250
300
350
400
20414
diagram: R. Gutell; http://www.rna.icmb.edu
CP111CP111
CP148CP148CP303CP303
![Page 10: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/10.jpg)
Circular permutation of the ribozymesequence changes the folding kinetics
0
0.2
0.4
0.6
0.8
1
0 10 20 30 40 50 60
fN
time (min)
CP111: 0.02 min-1
CP303: 3.1 min-1
wt: 0.39 min-1
Heilman-Miller & Woodson, J. Mol. Biol. 328, 385-394 (2003)
CP111CP111
CP148CP148
CP303CP303
![Page 11: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/11.jpg)
Circular permutation changes folding paths
Lease et al., J. Mol. Biol. 373, 197-210 (2007)
![Page 12: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/12.jpg)
Stable bacterial group I ribozyme
Adams et al., Nature 430, 45-50 (2004)
![Page 13: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/13.jpg)
Two Mg2+-dependent folding transitions
Rangan et al., PNAS 100: 1574-1579 (2003)
![Page 14: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/14.jpg)
Two Mg2+-dependent folding transitions
RNase T1 (2˚) •OH (3˚)
native gels activity
0
0.2
0.4
0.6
0.8
1
0
0.04
0.08
0.12
0.16
0.01 0.1 1 10
ƒN
[MgCl2] (mM)
kobs
(min-1)
Rangan et al., PNAS 100: 1574-1579 (2003)
![Page 15: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/15.jpg)
Two Mg2+-dependent folding transitions
Rangan et al., PNAS 100: 1574-1579 (2003)
![Page 16: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/16.jpg)
Ribozyme folds via native-like intermediates
backbone solvent
accessible
3˚? 3˚
![Page 17: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/17.jpg)
Loop-helix interaction stabilizes ICGUAA
Chauhan et al., J. Mol. Biol. 353, 1199-1209 (2005)
wtGUAA
30
35
40
45
50
55
60
65
0.01 0.1 1 10
0
0.1
0.2
0.3
0.4
0.5
0.6
Rg (
Å)
[Mg2+] (mM)
!U
U
IC
SAXS
![Page 18: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/18.jpg)
Ribozyme folds via native-like intermediates
Rangan et al., PNAS 100: 1574-1579 (2003)
Rg = 65 Å 30-32 Å 30 Å
3˚ 3˚
![Page 19: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/19.jpg)
Cations trigger rapid 3D collapse of RNA
Woodson, Curr. Opin. Chem. Biol. (2005)
![Page 20: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/20.jpg)
Specific coordination of Mg2+ by active site
Stahley & Strobel, Science 309, 1587-1590 (2005)
![Page 21: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/21.jpg)
Mg2+ localized by electrostatic field of RNA
Misra & Draper, J. Mol. Biol. 299: 813-825 (2000)Westhof & Sundaralingam, Biochemistry 25: 4865-4878 (1986)
![Page 22: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/22.jpg)
RNA more stable in multivalent cations
[Co(NH3)6]+3 > spd+3 > Mg+2 > Ba+2 > Na+ ~ K+
0
0.2
0.4
0.6
0.8
1
0.001 0.01 0.1 1 10 100 1000 104
ƒN
[Cation] (mM)
nH = 20
Heilman-Miller et al., J. Mol. Biol. 306,1157-1166 (2001)
![Page 23: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/23.jpg)
Stability of RNA depends on counterion size
Koculi et al., J. Mol. Biol. 341, 27-36 (2004); JACS 129, 2676-2682 (2007)
Folding of Tetrahymena ribozyme in divalent metals
![Page 24: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/24.jpg)
RNA less tightly folded in large cationsChange in dynamics?
mobility of RNA in PAGE depends on <r2>
Koculi et al., JACS 129, 2676-2682 (2007)
![Page 25: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/25.jpg)
Interactions between condensed ions
Small cations:
when s < Debye length, ionsinteract with each other
P
P
P
P
++
++
++
s
Large cations:
ions with large excludedvolume pack less efficientlyless charge neutralization
P
P
P
P ++ s
++
++
![Page 26: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/26.jpg)
Monovalent ions allow faster dynamicsHeilman-Miller et al.Figure 7
non-specificcollapse
Mg2+
extendedcompact
disorderednativeordered
non-specificcollapse
Mg2+
INS IN N
Monovalent cations:
Multivalent cations:
reordering
C+
fast
reordering
C+
slow
CC
CC
Cz+
C+
![Page 27: How RNA tells right from wrong: base pairs, tertiary ...€¦ · APS Argonne Soenke Siefert (BESSRC) Elena Kondrashkina Jaby Jacob (BioCAT) NSLS Brookhaven Mike Sullivan Sayan Gupta](https://reader033.fdocuments.in/reader033/viewer/2022050408/5f84fba9b190d966d926220f/html5/thumbnails/27.jpg)
Acknowledgements
Woodson lab
Gokhan Caliskan
Seema Chauhan
Susan Heilman-Miller
Scott Jackson
Eda Koculi
Sujatha Koduvayur
Tania NikolchevaJie Pan
Priya Ramaswamy
Prashanth Rangan
Adi Tadepalli
Funding NIH, NIST
APS Argonne
Soenke Siefert(BESSRC)
Elena Kondrashkina
Jaby Jacob(BioCAT)
NSLS BrookhavenMike SullivanSayan Gupta
AECOMMichael Brenowitz
Bianca Sclavi
U. Rochester
Gloria Culver
University of Maryland
D. Thirumalai
Rob Briber
NIST Gaithersburg
Ursula Perez-Salas
Susan Krueger
IMBC-Strasbourg
Benoît Masquida
Eric Westhof
JHMI
Rachel Green
Anthony Cukras