Post on 22-Feb-2016
description
RNA catalysis
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 ribozymehammerhead 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 splicinggroup I introns (crystal structure)group II introns*** U2-U6 snRNA (spliceosome) (partial NMR structure) ***
• Peptide bond formationribosome (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 self-
cleaving (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 AProtein enzyme
Hammerheadribozyme
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 sessile 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 sessile phophodiester 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 showssubstrate positioningaccounts for catalysis
Found 36 times in rRNA as type II/I couples
Numerous isolated type I interactions
Prevalence of A-minor motifs
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 molecules isolated from in vitro selection1
Catalytic Nucleic Acid Reaction Catalyzed or Activity
RNA Aminoacyl esteraseRNA DNA CleavageRNA RNA CleavageRNA RNA LigationRNA Isomerization of a bridged biphenylRNA Self-phosphorylationRNA Amide bond cleavageRNA AminoacylationRNA AlkylationRNA 5'-5' RNA ligationRNA Acyl transferase (ester and amide bond formation)RNA Porphyrin metalation with Cu2+ (heme biosynthesis)RNA Sulfur alkylationRNA 5'-self-cappingRNA Carbon-carbon bond formation (Diels-Alder cycloaddition)RNA Amide bond formationRNA Peptide bond formationRNA Ester transferase
DNA RNA cleavageDNA DNA ligationDNA Porphyrin metalation with Cu2+ (heme biosynthesis)DNA Cleave phosphoramidate bondsDNA DNA cleavageDNA Self-phosphorylationDNA 5'-self-capping
1Ref. 44. This list is only an overview and does not include all nucleic acid catalystsdiscovered to date.
DNA Catalysts
DNA CatalystsGuanine Quartet Structures
HDV ribozyme structure
Proposed mechanism of catalysis
pH (pD) profiles
pH profiles (cation type)
pH profiles (cation concentration)