Improved Thermodynamic Model for Predicting RNA 3 × 3 Internal Loops and NMR Structure of an Unusually Stable Loop with Three Consecutive Sheared GA Pairs Gang Chen, Brent M. Znosko, Scott D. Kennedy, Xiaoqi Jiao, Thomas R. Krugh, Douglas H. Turner Department of Chemistry, University of Rochester, NY 14627-0216 NMR STUDIES: NMR structures provide insight into the interactions that determine stability and also provide a start for prediction of three dimensional structure from sequence.

Goal of Turner group Predict RNA secondary structure for up to 1000 nucleotides with accuracy of 85%, using Free Energy Minimization and experimental constraints. Unlike protein, the interactions determining RNA secondary structure, primarily hydrogen bonding and base stacking, dominate over those of tertiary structure. Thus studies (e.g. UV melting and NMR) of short oligonucleotides (~10 base pairs) can provide insight into folding of large RNAs.

The unusually stable, relatively abundant and functionally important motif: NMR construct:

Why 3 × 3 internal loop? ---- Middle pair can both stabilize and destabilize the loop Internal loops are important motifs for tertiary interactions and ligand binding.

GGA AAG

GGA AAG

∆G°37 kcal/mol

Kd µM

-13.3

4.2×10-4

60.8

-2.6

-13.8

1.9×10-4 64.4

-2.2

-11.9

4.1×10-3

-0.4

motif in LSU (left) and SSU (right) rRNA [2,3]

Tm ∆G°37, loop °C kcal/mol

59.1

P = Purine riboside

Predicting the sequence dependence of folding stability and structure of internal loops is at an early stage. The current RNAstructure 4.1 program only considers closing base pairs and loop-terminal pairs.

1D imino proton NMR spectra at various temperatures in H2O

SNOESY spectrum at 5 °C in H2O

“Walk region” of NOESY spectrum with mixing time 400 ms at 30 °C in D2O

HSQC (H1’-C1’ and H5-C5 region) at 30 °C

3 × 3 internal loops are the smallest loop with one potential non-canonical base pair flanked by a noncanonical base pair on both sides, and the identity of the middle pair affects stability. [1] 6H2-15H1’

5′CGCGAAGGC3′ +

3′GCGAAGCCG5′ 5′CGCGAAGGC3′ +

3′GCGAGGCCG5′

5′CGCGAAGGC3′ 3′GCGAAGCCG5′

∆G°37 kcal/mol

Kd µM

Tm °C

-8.4

1.2

46.5 CG: Closing base pair

15H2-6H1’

C1’

Cytosine C5

15H2-16H1’

Uridine C5

GA: Loop-terminal pair

5′CGCGAAGGC3′ 3′GCGAGGCCG5′

5.5×10-2

-10.3

54.3

AA/GA: Middle pair Superposition of 30 low-energy structures derived from NMR restrained molecular dynamics and energy minization

Stereo view of one of the modeled low-energy structures

Solvent accessible surface of major (left) and minor (right) groove.

Comparison between RNAstructure 4.1 and model developed here for predicting 3 × 3 internal loops carbon oxygen nitrogen hydrogen

3 RNAstructure 4.1

Entropy penalty

Closing base pair

Loop-terminal pair

phosphorus stem residues

Measured (kcal/mol)

2

1

∆G˚predicted = ∆G˚loop initiation + ∆G˚AU penalty + ∆G˚UU bonus + ∆G˚GA bonus + ∆G˚AG bonus

0

-1

R2: 0.29 σ: 0.85 kcal/mol

kcal/mol

1.9 ± 0.1

0.7 ± 0.05

-0.7 ± 0.1

-1.0 ± 0.1

-0.8 ± 0.1

-2 -2

-1

0

1

2

3

Middle pair

Base stacking of three consecutive sheared GA pairs

Potential hydrogen bonding network involving backbone groups within the loop

Base-base hydrogen bonds for standard wobble GU and sheared GA pairs

3

Fit to bottom eq

G4A16

Measured (kcal/mol)

2

G5A15

∆G˚predicted = ∆G˚loop initiation + ∆G˚AU penalty + ∆G˚UU bonus + ∆G˚GA bonus + ∆G˚AG bonus + ∆G˚middle bonus + ∆G˚5’GU/3’AN

1

O P O

O P O

5' N

N

O

R2: 0.76 σ: 0.49 kcal/mol

0.84 ± 0.15

-0.49 ± 0.11

-0.89 ± 0.09

-0.37 ± 0.14

-1.22 ± 0.14

0.95 ± 0.22

1

2

H2N

Predicted (kcal/mol)

Acknowledgment: Thanks to Prof. R. Kierzek, Prof. Dave Mathews, Dr. Matt Disney, Dr. Jessica Childs, Dr. Sandip Sur, and all Turner lab members. NIH grant GM 22939

For loops with a single loop-terminal GA pair that has a U 3’ to the G of the GA pair

G7

H

N

HO

3'

G7C13 (closer to viewer)

G14

G

N

N

O

N

H

H

H

H

N N

N N

H N

H

A N

O

N

R

N

O P O

H

R

N

H

O

N/S

O P O

H

N

R

OH

O

N

O P O

H

O

C13

HO

O

O P O

R

HN A15

OH

N

H U

H2N H2N

A16

N

For a GA middle pair flanked by at least one non-pyrimidine-pyrimidine loop-terminal pair

G

O

H2N

NH

NH2

G17

3

A6

G5

NH

O

0

OH

OH

NH2

HO

-1

H

N

N

-2 -2

OH

G4 H2N

1.99 ± 0.11

N H

O

N

0

kcal/mol

O

N

O

OH

U3

-1

N/S?

O

OH

3'

O P O

S

O

A6G14 (closest to viewer)

O P O

5'

U3G17 (farther to viewer)

References: 1.

Chen, G., Znosko, B. M., Jiao, X., and Turner, D. H. (2004) Factors affecting thermodynamic stabilities of RNA 3 × 3 internal loops, Biochemistry In press.

2.

Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Structure of the 30S ribosomal subunit, Nature 407, 327-339.

3.

Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000) The complete atomic structure of the large ribosomal subunit at 2.4 angstrom resolution, Science 289, 905-920.

H