RNA catalysis
Understand the basics of RNA/DNA catalysts - what functional
groups used for catalysis? structures formed?
Know about transesterification & cleavage reactions
Know four types of natural catalytic RNAs (group I introns, group II
introns, RNase P, small self-cleaving), what reactions they perform,
know basics of their secondary and tertiary structure, requirements
for cofactors/metals/proteins/ATP
Know details of glmS ribozyme self-cleavage
Understand use of ribozymes as therapeutics
In vitro selection - understand the process
Know some of the ribozymes and deoxyribozymes that have been
discovered using in vitro selection
Outline
•
RNA transesterification
•
Naturally occurring catalysts
•
Catalytic functions
•
Catalytic mechanisms
RNA transesterification
•
Exchange one phosphate ester for another
•
Free energy change is minimal (reversible)
RNA transesterification
•
Nucleophile can be either the adjacent 2´ hydroxyl or
another ester
•
Referred to as hydrolysis when water serves as the
nucleophile
RNA transesterification
•
Nucleophilic attack on the phosphorus center leads to a
penta-coordinate intermediate
•
Ester opposite from the nucleophile serves as the leaving
group (in-line attack)
General mechanisms
•
Substrate positioning
•
Transition state stabilization
•
Acid-base catalysis
•
Metal ion catalysis
RNA Catalysts
Naturally occurring catalysts
•
RNA cleavage
glmS ribozyme (crystal structure)
hammerhead ribozyme (crystal structure)
hairpin ribozyme (crystal structure)
Varkud satellite (VS) ribozyme (partial NMR structure)
hepatitis delta virus (HDV) ribozyme (crystal structure)
M1 RNA (RNase P) (partial crystal structure)
•
RNA splicing
group I introns (crystal structure)
group II introns (crystal structure)
*** U2-U6 snRNA (spliceosome) (partial NMR structure) ***
•
Peptide bond formation
ribosome (crystal structure)
Small self-cleaving ribozymes
•
Hammerhead, hairpin, VS, HDV ribozymes
•
Derivative of viral, viroid, or satellite RNAs
•
Involved in RNA processing during rolling circle
replication
•
RNA transesterification via 2´ hydroxyl
•
Reversible: cleavage and ligation (excepting HDV)
Hammerhead ribozyme
•
Three-stem junction with conserved loop regions
•
Coaxial stacking of stems II and III through
extended stem II structure containing canonical
Watson-Crick and non-canonical base pairs
•
Metal-ion catalysis
Hammerhead ribozyme
•
In nature is selfcleaving (not a true
enzyme)
•
Can be manipulated
to function as a true
catalyst
•
Biotechnological and
potential therapeutic
applications for
target RNA cleavage
Hammerhead ribozyme
• Separation of catalytic and substrate strands
• Strand with hairpin is the enzyme
• Single strand is substrate
• KM = 40nM; kcat = ~1 min-1;
kcat/KM = ~107 M -1 min -1 (catalytic efficiency)
• Compare to protein enzymes?
RNA Catalysts
• basics of catalytic reactions (cleavage)
RNase A
Protein enzyme
Hammerhead
ribozyme
Hairpin ribozyme
•
In nature is part of a four-stem junction
•
Ribozyme consists of two stems with internal loops
•
Stems align side-by-side with 180 degree bend in
the junction (hence ‘hairpin’)
•
Internal loops interact to form active site
Hairpin ribozyme
•
Crystal structure
reveals interactions
between stems
•
Nucleobases position
and activate scissile
phosphodiester
linkage
•
Combination of
transition state
stabilization and
acid-base catalysis?
HDV ribozyme
•
Genomic and
antigenomic
ribozymes
•
Nested
pseudoknot
structure
•
Very stable
•
Cleaves off 5´
leader sequence
HDV ribozyme
HDV ribozyme
•
Active site positions an
important cytidine near
the scissile
phosphodiester bond
RNase P
•
True enzyme
•
Cleaves tRNA precursor to
generate the mature 5´ end
•
Composed of M1 RNA and C5
protein (14 kD)
•
RNA is large and structurally
complex
•
Protein improves turnover
•
Hydrolysis
Group I introns
•
Large family of self-splicing introns usually
residing in rRNA and tRNA
•
Two step reaction mechanism
Group I intron structure
•
Crystal structure of ‘trapped’
ribozyme before second
transesterification reaction
•
Metal ion catalysis
Group I intron structure
Ribose zipper
P1
J8/7
Group II introns
Group II introns
•
Usually found in organelles
(e.g. plant chloroplasts,
mitochondria)
•
mechanism proceeds through a
branched lariat intermediate
structure which is produced by
the attack of a 2’-OH of an
internal A on the
phosphodiester of the 5’-splice
site
•
proteins thought to stabilize
structure but not necessary for
catalysis
•
no ATP or exogenous G needed
Summary of splicing reactions
The ribosome is a ribozyme
•
Ribosome is 2/3 RNA and 1/3 protein by mass
•
Crystal structures prove that RNA is responsible for
decoding and for peptide bond formation
Peptidyl transferase
• Crystal structure of 50S subunit shows no protein within 20 Å
of peptidyl transferase center
• Closest component to
aa-tRNA is adenosine
2451 in 23S rRNA
• Proposed acid-base
mechanism for peptide
bond formation
• Recent evidence shows
substrate positioning
accounts for catalysis
Glucosamine 6-phosphate riboswitch/ribozyme
•
Glucosamine-6-phosphate
(GlcN6P)-dependent selfcleaving ribozyme
•
Regulates biosynthesis of
amino sugars used in
bacterial cell wall synthesis
glmS is a metabolite-responsive ribozyme
Effects of [glcN6P] on the rate constant.
M
Optimization of catalysis by the glmS ribozyme
Glucosamine 6-phosphate ribozyme self-cleavage
RNA transesterification
Glucosamine 6-phosphate ribozyme self-cleavage
RNA transesterification
Might glucosamine 6-phosphate serve as the
general acid-base (coenzyme) for self-cleavage?
Ribozyme exhibits self-cleavage activity in
TRIS buffer in the absence of ligand
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A.,
Soukup, J.K. & Soukup, G.A. "Ligand
requirements for glmS ribozyme self-cleavage."
Chemistry & Biology 12:1-6 (2005).
Ligand specificity importance of amine functionality
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand
requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12:1-6 (2005).
Observed rate constants and apparent binding of ligand analogs
Table 1. Kinetic parameters for the glmS ribozyme in the absence or presence of 10 mM
GlcN6P or various analogs.
ligand
kobs (min-1)
appa rent KD (mM)
rate enhance ment
GlcN6P
GlcN
Serinol
1.1
3.0 x 10-2
7.5 x 10-3
0.03
³5
³5
110,000
3,000
750
TRIS
Π
1.3 x 10-3
~10-5
³25
Π
130
Π
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand
requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12:1-6 (2005).
pH-reactivity profiles
•
GlcN and serinol are lower
affinity ligands
•
Apparent pKa for liganddependent self-cleavage
approximates the solution pKa of
ligand
•
Suggest the amine functionality of
the ligand functions as a general
acid/base in catalysis
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand
requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12:1-6 (2005).
RNA/DNA Catalysts
RNA/DNA catalysis & evolution
• in vitro selection
RNA/DNA Catalysts
RNA/DNA catalysis & evolution
• increasing numbers of examples of reactions catalyzed by nucleic acids
Table 1. Catalytic RNA and DNA mo le cules isolated from in vitro selection1
Catalytic Nucleic Acid
1
Reaction Catalyze d or A ctivity
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
Aminoacyl esterase
DNA Cleavage
RNA C lea vage
RNA Ligation
Isomerization of a bridged biphenyl
Self-phosphorylation
Amide bond cleavage
Aminoacylation
Alk ylation
5'-5' RNA ligation
Acyl transferase (ester and amide bond formation)
Porphyrin metalation with Cu 2+ (heme biosynthesis)
Sulfur alkylation
5'-self-cappin g
Carbon-carbon bond formation (Diels-Alder cycloaddition)
Amide bond formation
Peptide bond formation
Ester transferase
DNA
DNA
DNA
DNA
DNA
DNA
DNA
RNA cleavage
DNA ligation
Porphyrin metalation with Cu 2+ (heme biosynthesis)
Cleave phosphoramidate bonds
DNA cleavage
Self-phosphorylation
5'-self-cappin g
Ref. 44. Th is li st is only an overv ie w and does no t includ e all nuc leic acid catalysts
discover ed to date.
DNA Catalysts