HAMMERHEAD RIBOZYME STRUCTURE VIA SITE-DIRECTED SPIN-LABELING AND

EPR/DEER SPECTROSCOPY

Fanshon MontgomeryAlcorn State UniversitySPUR Program 2009

RNA

What is RNA?

The Structure

RNA vs DNA

DNA is double stranded. RNA is usually single stranded.

http://www.phschool.com/science/biology_place/biocoach/transcription/chains.html

RNA SPLICING Central Dogma

of Molecular Biology

Types of RNA Splicing in RNA

tRNA rRNA snRNA miRNA siRNA tmRNA piRNA Viral RNAhttp://www.phschool.com/science/biology_place/biocoach/transc

ription/premrna.html

RIBOZYMES

Discovered in 1982.

Different Types of Ribozymes

The Hammerhead ribozyme (HHRz) is a self-cleaving RNA motif important to replication of plant viroids.

VIROIDS

www.ars.usda.gov/pandocs.htm

Small RNA agents of infectious plant diseases

Rolling Circle Replication

III

I II

L1 L2

Viroid

RNA

cleave

gene copy

SCHISTOSOMA MANSONI HHRz motifs

with unknown function are predicted in several other genomes.

Sequence derived from Schistosoma mansoni

www.usuhs.mil/mic/Davies/Research.html

OVERALL GOAL

Use SDSL to show how the docked and active forms of the Hammerhead Ribozyme differ in structure to give insight about the cleavage (splicing) mechanism.

MMg2+M

activeMg2+

PREVIOUS RESEARCH Previous kinetics

measurements, dynamic studies, and distance measurements have been vital to the investigation of catalytic activity in the HHRz.

EPR and Deer Measurements

L2.1U1.6

U1.6L2.1 L2.1 U1.6L2.1

Kim, Ayaluru, DeRose, JACS 2005

CURRENT RESEARCH GOALS

Predictions: 1. U7 and U1.6 will

get closer in active form.

2. U1.6 and L2.1 will stay the same in active form.

Mg2+ Mg2+M M active

CL2.1 U1.6

U7

CL2.1CL2.1 U1.6U1.6

U7 U7

SPIN-LABELING REACTION

Spin-Labeling Mechanism-4-isocyanato TEMPO with a 2'-amine-modified uridine

NH

O

ON

O

NH2O

HH

HH

PO

O

O

O-

NO

NC

O

NH

O

ON

O

HNO

HH

HH

PO

O

O

O-

NO

NC

-O

NH

O

ON

O

HNO

HH

HH

PO

O

O

O-

NO

NC

-O

NH

O

ON

O

HNO

HH

HH

PO

O

O

O-

NO

HNC

O

- H+

+ H+

10-MERS We used Pulsed EPR to determine the

distance between spin-labels on the Hammerhead Ribozyme. These 10-mers were used a proof of concept through the previous graduate student’s molecular modeling. They gave us a good idea of how far apart the spin labels on the 10-mers actually are.

1. U5/U7A- 2. U3/U7-5’CCUAGUGUGG3’ 5’CCUAUGGUGG3’3’GGAUACCACC5’ 3’GGAUCACACC5’ 3. U8/U7- 5’CCUAGUGUGG3’ 3’GGAUCACACC5’

CURRENT RESEARCH GOALSLarger RNA- 43-mers

L2.1

7

PURIFICATION OF SPIN-LABELED RNA

Mass Spectrometry- Confirmed product formation

HPLC-Purification and separation

Unlabeled RNA

m/z 6294

Separated Spin-label m/z 6463

CONTINUOUS WAVE EPR

EPR Spectroscopy-Detects chemical species that have unpaired electrons.

PULSED EPR/DEER SPECTROSCOPY

Double Electron Electron Spectroscopy-Determines the distances between spin-labels

Bowman, M. K.; Maryasov, A. G.; Kim, N.; DeRose, V. J. Appl. Magn. Reson. 2004, 26, 23-39.

PULSED EPR/DEER SPECTROSCOPY

U7/U3 Echo Field Sweep

Field (Gauss)

3400 3450 3500 3550

EPR

Inten

sity

0

1e+5

2e+5

3e+5

4e+5

5e+5

6e+5

U7/U3 Echo Decay

Time (ns)

-200 0 200 400 600 800 1000

DEER

(T)

9.0e+6

1.0e+7

1.1e+7

1.2e+7

1.3e+7

1.4e+7

1.5e+7

CONCLUSIONS

1. Spin- Labeling Successful-8 strands of RNA-purified and isolated RNA

2. EPR/DEER Analysis-confirmed spin-labeled-DEER spectra-weak signal-to-

noise ratio

FUTURE WORK

Main Goal: To redo DEER experiments with

more sample. This could lead to great insight about the folding mechanism of the HHRz.

Acknowledgements I would like to generously thank the following

for the privilege and opportunity to be apart of this research!

Dr. Victoria DeRose Dr. Brandon Green The DeRose Laboratory William Byrant Adam Unger NSF NIH SPUR Program-Peter O’Day The University of Washington-Stefan

Ochsenbien