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 ribozyme 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 *** 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 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? Proteins typically have much greater kcat and much higher Km. The 40 nM Km of the hamemrhead indicates an extremely stable enzyme-substrate complex which will not dissociate product readily. This slow release of product is the reason for the low kcat. A protein enzyme with a Km/kcat of 10-7 might have a kcat of 105/min and Km of 10-2M

RNA Catalysts RNase A Protein enzyme Hammerhead ribozyme • 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 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 shows substrate positioning accounts for catalysis

Prevalence of A-minor motifs Found 36 times in rRNA as type II/I couples Numerous isolated type I interactions

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

DNA Catalysts

Guanine Quartet Structures DNA Catalysts Guanine Quartet Structures

HDV ribozyme structure

Proposed mechanism of catalysis

pH (pD) profiles

pH profiles (cation type)

pH profiles (cation concentration)