Download A G--C

RNA Structure Prediction
RNA Structure Basics
The RNA ‘Rules’
Programs and Predictions
Assigned reading: Ch. 6 from Bioinformatics: A Practical
Guide to the Analysis of Genes and Proteins, 3rd Ed. by
Baxevanis and Ouellette.
BIO520 Bioinformatics
Jim Lund
RNA classes
• mRNA - messenger RNA.
• tRNA - transfer RNA, small (~80 bases) sequences
which bring amino acids to the ribosome.
• rRNA - ribosomal RNA, RNA + proteins =
ribosome.
• viral RNA (ssRNA, dsRNA virii)
• miRNA: translational/transcriptional gene silencing.
• snoRNA, snRNA: splicing, RNA bp modification
• Transfer-Messenger RNAs (tmRNA), Small
cytosolic RNAs (scRNA), Guide RNAs (gRNA)
• and more…
RNA structures
• 1°
– Sequence (and modifications)
• 2°
– Base pairing
• 3°
– Overall Structure, non Watson-Crick
pairs
– Experimental structures: tRNA,
ribosome
RNA Tertiary Structure, tRNA
3’(aminoacyl) end
CCA
Anticodon Loop
Yeast Phenylalanine tRNA, 1.93A
Yeast Phenylalanine tRNA, 1.93A
rRNA small subunit, X. laevis
2° RNA structures
• Watson-Crick pairing -> helices
• Loop regions
–
–
–
–
Hairpin loops
Internal loops
Bulge loops
Multibranch loops
RNA Modifications
Covalent Modifications-especially tRNA
–
–
–
–
rUrT, r, rD, rS4U
rC 3-CH3-C, 5-CH3-C
rA I, 6-CH3-A, 6-isopentenyl-A
rG  7-CH3-G, Q, Y
Nucleosides Nucleotides 1999 Jun-Jul;18(6-7):1579-81
RNA Base pairing
• G-C triple hydrogen bond
• A -U double hydrogen bond
• G-U single hydrogen bond
RNA structure energetics
• The number of GC versus AU and GU base
pairs.
– Higher energy bonds form more stable structures.
• Number of base pairs in a stem region.
– Longer stems result in more bonds.
• Number of base pairs in a hairpin loop region.
– Formation of loops with more than 10 or less than 5
bases requires more energy.
• Number of unpaired bases (interior loops or
bulges).
– Unpaired bases decrease the stability of the structure.
2° Structure
5’
3’
G--C
G--C
C--G
A
|
U--A
G--C
A
A
A
A
A A
“The Rules”
• Base Pairs -- Good
– G:C better than A:T -- And local sequence
matters!
• Bulges, Loops -- Bad
• Many small interactions---Stable
Structure
• Only predict “Canonical Interactions”
Base Pairs/Stacks
A
A
U
U
Basepair
A=U
A=U
G = -1.2 kcal/mole
A
U
U
A
Basepair
A=U
U=A
G = -1.6 kcal/mole
Base Pairing/Stacking
AA
UU
-1.2
CG
GC
-3.0
AU or UA
UA
AU
-1.6
GC
CG
-4.3
AG, AC, CA, GA
UC, UG, GU, CU
-2.1
GU
UG
-0.3
CC
GG
-4.8
XG, GX
YU, UY
0
Bloomfield, Crothers, Tinoco, Physical Chemistry of Nucleic Acids
Hairpin Loops
(GC closure)
N=3
+8
N=4,5
+5
N=6,7
+4
N=8,9
+5
N>=10
6+0.9(ln[N/10])
•Tertiary Interactions!
Internal Loops
5’
3’
G--C
G--C
C--G
A
G
G
G-X-C
C-X-G
N=2-6
+2
N=7
+3
N>=8
3+0.9(ln[N/7])
0
A
A
C
T--A
G--C
T--A
G--C
Single-Strand Bulges
5’
3’
G--C
G--C
C--G
A
|
G
|
A
|
T--A
G--C
T--A
G--C
N=1
+3
N=2-3
+4
N=4-7
+5
N>=8
6+0.9ln(N/8)
Prediction Programs
• Mfold (M. Zuker)
– 2° structure
• RNAstructure/OligoWalk
– 2° structure, oligo/RNA target interactions
• alifold
– 2° structure constrained by muliple
alignment.
• Pfold
– 2° structure guided by rules derived from
known tRNA/rRNA structures
Prediction Programs
• Mfold (GCG)
– M. Zuker
• Mfold input to Plotfold
– Non-graphic output -G option
– Graphics outputs
•
•
•
•
•
SQUIGGLES
mountains
circles
domes
energy plots
Squiggles
1
CCA-3’OH
60
20
40
DOMES, MOUNTAIN, CIRCLES
MFOLDStructure Family
• Optimal & Suboptimal structures
– Can ask for multiple structures
• Energy increment and “window size”
increment.
• View individually.
• How variable are the structures?
– Energy Plots
ENERGY PLOT
P-Num Plot
Prediction Quality
Forces in RNA folds
• Complementary molecular surfaces
• Bridging cations
• Pseudoknotting
• “kinetic traps” in folding
– NOT always 2 first!
Annu Rev Biophys Biomol Struct 1999;28:57-73
Proc Natl Acad Sci U S A 1998 Sep 29;95(20):11555-60
RNA Structure Probing
• Physical methods
– X-ray diffraction, NMR
• Enzymatic methods
– S1, Rnases (find ss and ds regions).
• Chemical modification
– DMS…
• Mutagenesis
– G:C=>C:C=>C:G
Ribozymes
• Naturally occurring
–
–
–
–
RNAaseP
Group I introns
Group II introns
snRNA in the splicosome
• Artifical
– Engineered/evolved in the lab from natural
ribozymes to have new substrate RNA.
– Cleave mRNA, drug-like action
• miRNA/siRNA
– Translational/transcriptional gene silencing
Cross-replicating RNA enzymes
Published by AAAS
T. A. Lincoln et al., Science 323, 1229 -1232 (2009)