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RNA structure can be specific, stable and complex.(As a result, RNA mediates specific recognition and catalytic reactions.)
Principles/ideas--RNAs contain characteristic 2° and 3° motifsSecondary structure--stems, bulges & loops
Coaxial stackingMetal ion binding
Tertiary motifs (Pseudoknots, A-A platform, tetraloop/tetraloop receptor,A-minor motif, ribose zipper)
Patterns and principles of RNA structure
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RNA vs. DNA
nucleoside
nucleotide
glycosidicbond
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RNA vs. DNA: who cares?
Base-catalyzed RNA cleavage!
-OH
Stablebackbone
Unstablebackbone
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RNA transesterification mechanism
Base-catalyzed RNA cleavage!
transition state
++
-OH
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Different bases in RNA and DNA
RNAonly
DNAonly DNA and RNA
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RNA chain is made single stranded!
Chain is directional. Convention: 5’ 3’.
Chemical schematic One-letter code
ssDNA can signal DNA damage and promote cell death
dsRNA canblock proteinsynthesis and signal viralinfections
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Six backbone dihedral angles per nucleotide in RNA and DNA
Is ssDNA floppy or rigid? Is RNA more or less flexible than ssDNA?
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Two orientations of the bases: Anti and syn
DNA and RNA
Absent fromundamageddsDNA
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-OH, what a difference an O makes!
Different functions of DNA and RNA
Stores genetic info Stores genetic infossDNA signals cell death ssRNA OK
E.g. mRNA = gene copydsDNA OK dsRNA (“A” form) signals infection,
mediates editing, RNA interference,. . .
Double helical (B form) Forms complex structuresSupercoiled Enzymes (e.g. ribosome),
Binding sites & scaffoldsSignalsTemplates (e.g. telomeres)
gene1gene2
gene3 . . .
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Examples of RNA structural motifsStem, bulge, loop 4-helix junction TetraloopPseudoknotSheared AA pairsPurine stacksMetal binding sitesA-A platformTetraloop receptorA-minor motifRibose zipper . . .
Secondary structures Tertiary structures
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Cloverleaf representation of yeast Phe tRNA
Coaxial stacking of adjacent stems forms an L-shaped fold
“Cloverleaf” conservedin all tRNAs
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Schematic drawing of yeast Phe tRNA fold
Mg2+ (balls)
Spermine
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Non-WC base pairs and base triples in yeast tRNA Phe
LOTS OF BASE COMBOS!! Enable alternate backbone orientations:
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A9 intercalates between adjacent G45 and m7G46 in yeast tRNA Phe
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Examples of RNA structural motifs
TetraloopPseudoknot4-helix junctionSheared AA pairsPurine stacksMetal binding sitesA-A platformTetraloop receptorA-minor motif . . .
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UNCG tetraloop
Stabilizes attached stem
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HIV TAR RNA mediates Tat binding2° structure schematic
Nomenclature for secondarystructure: stem, loop & bulge
Coaxial stacking
Basetriple Arg binds
GC bp
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HIV TAR RNA mediates Tat binding2° structure schematic
Nomenclature for secondarystructure: stem, loop & bulge
Coaxial stacking
Basetriple
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HIV TAR RNA mediates Tat binding2° structure schematic
Nomenclature for secondarystructure: stem, loop & bulge
Coaxial stacking
Basetriple Arg binds
G26/C39 bp
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PseudoknotsHDV ribozyme forms a double pseudoknot
Bases in loop of stem 1 form stem
2 (with bases outside stem 1)
1
2
1
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Hepatitis Delta Virus (HDV) ribozyme double pseudoknot
“Top” view
U1A protein cocrystals2° structureschematic
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Hepatitis Delta Virus (HDV) ribozyme double pseudoknot
“Top” view
U1A protein cocrystals2° structureschematic
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Four-helix junction: L11 protein binding site in 23S RNA
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Four-helix junction: L11 protein binding site in 23S RNA
Four helices emerge from a central wheel.The four double-helical stems form two coaxial stacks.The two stacks have irregular but complementary shapes.The helices knit together to form a compact globular domain.
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Base triples in the L11 4-helix junction
Bulge and loop mediate long-range tertiary interactions.The riboses of A1084-A1086 (all A’s) form a “ribose zipper.A1086 adopts a syn conformation to facilitate tight sugar packing.
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Metal ions stabilize the L11 RNA 4-helix junction
Mg2+ ions (gold balls)Cd2+ ions (magenta)Hg2+ (rose)
RNA interactions ofthe central Cd2+ ion
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P4-P6 Domain of the Group I ribozyme
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P4-P6 Domain of the Group I ribozyme
Two helical stacks are arranged parallel to each other. The structure is one helical radius thick.Two regions of 3° interactions between the two helical stacks.
1. Tetraloop/Tetraloop-receptor.2. A-rich, single-stranded loop and the minor groove of the
opposing helix.
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Tertiary interactions in the P4-P6 domain
Cross-strand purine stack.
Sheared AA Standard AU
Sheared AA bps fill minor groove
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Tertiary interactions in the P4-P6 domain
Side view
A-A platformAdjacent As pair side-by-
side
Top view
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Tertiary interactions in the P4-P6 domain
Side view
A-A platformAdjacent As pair side-by-
side
Top view
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Tertiary interactions in the P4-P6 domain
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Tertiary interactions in the P4-P6 domain
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Tertiary interactions in the P4-P6 domain
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Metal ion core in the P4-P6 domain
Divalent metal ions (Mg2+) are required for proper folding.
These ions bind to specific sites and mediate the close approach of the phosphate backbones
At one position in the molecule the phosphate backbone turns inward and coordinates two metal ions.
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Adenosine-minor-groove base triples: the A-minor motif
A fills minor groove & ribose 2’ OH forms H-bonds
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Hydrogen bonds between adjacent backboneatoms create a “ribose zipper”
Adjacent base-triples bring together RNA strands
Deoxynucleotides destabilize P4-P6
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The A-minor motif is widespreadConserved As are abundant in unpaired regions of structured RNAs.
Group I intron
P4-P6
% of As in “single-stranded regions
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What happens in very large RNAs?
% of As in “single-stranded regions
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A-minor motifs are the predominant tertiary interaction in the 50S ribosomal subunit
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Summary1. RNA structure can be specific, globular, stable
and complex. (As a result, RNA mediates specific recognition and catalytic reactions.)
2. Secondary structures include stems, bulges, and loops.
3. Tertiary motifs include base triples, pseudoknots, A-A platforms, the tetraloop/tetraloop receptor,A-minor motifs, ribose zippers
4. Principles: stems and loops conserved, many non-WC base contacts, coaxial stacking, metal ion binding, H-bonding of ribose 2’ OH, and repeated “motifs”.