How RNA tells right from wrong:base pairs, tertiary interactions

and counterions

Sarah WoodsonJohns Hopkins University

folding

10-100 ms

transcription

25 nt/s

hairpins10-100 !s

folding

10-100 ms

misfolded

intermediate

slow

Cations trigger rapid 3D collapse of RNA

Woodson, Curr. Opin. Chem. Biol. (2005)

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

Ribozyme core misfolds

Guo et al., Mol Cell (2004) Sclavi et al, Science; Pan et al., JMB (1998)

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)

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

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

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

Circular permutation changes folding paths

Lease et al., J. Mol. Biol. 373, 197-210 (2007)

Stable bacterial group I ribozyme

Adams et al., Nature 430, 45-50 (2004)

Two Mg2+-dependent folding transitions

Rangan et al., PNAS 100: 1574-1579 (2003)

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)

Two Mg2+-dependent folding transitions

Rangan et al., PNAS 100: 1574-1579 (2003)

Ribozyme folds via native-like intermediates

backbone solvent

accessible

3˚? 3˚

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

Ribozyme folds via native-like intermediates

Rangan et al., PNAS 100: 1574-1579 (2003)

Rg = 65 Å 30-32 Å 30 Å

3˚ 3˚

Cations trigger rapid 3D collapse of RNA

Woodson, Curr. Opin. Chem. Biol. (2005)

Specific coordination of Mg2+ by active site

Stahley & Strobel, Science 309, 1587-1590 (2005)

Mg2+ localized by electrostatic field of RNA

Misra & Draper, J. Mol. Biol. 299: 813-825 (2000)Westhof & Sundaralingam, Biochemistry 25: 4865-4878 (1986)

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)

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

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)

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

++

++

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+

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