fanshon montgomery alcorn state university spur program 2009
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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’
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
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.
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