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

K

M

k

cat = 40nM; k /K M = ~10 7 cat = ~1 min -1 ; 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 sessile phosphodiester linkage Combination of transition state stabilization and acid-base catalysis?

• • • • Genomic and antigenomic ribozymes Nested pseudoknot structure Very stable Cleaves off 5´ leader sequence HDV ribozyme

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

• in vitro selection

RNA/DNA Catalysts

RNA/DNA catalysis & evolution

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

selection 1

Catalytic Nucleic Acid 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-phosphoryla tion Amide bond cleavage Aminoacyla tion Alk ylation 5'-5' RNA ligation Acyl transferase (ester and amide bond formation) Porphyrin metalation with Cu 2+ (heme biosynthesis) Sulfur alkyla tion 5'-self-c appin g Carbon-carbon bond formation (Diels-Alder cycloaddition) Amide bond formation Peptide bond formation Ester transferase DNA discover ed to date.

RNA cleavage DNA DNA DNA DNA DNA DNA ligation Porphyrin metalation with Cu 2+ (heme biosynthesis) Cleave phosphoramidate bonds DNA cleavage Self-phosphoryla tion DNA 5'-self-c appin g 1 Ref. 44. Th is li st is only an overv ie w and does no t includ e all nuc leic acid catalysts

DNA Catalysts

DNA Catalysts Guanine Quartet Structures

HDV ribozyme structure

Proposed mechanism of catalysis

pH (pD) profiles

pH profiles (cation type)

pH profiles (cation concentration)