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Approaches to understanding catalysis by RNA

Phosphodiester bond rearrangements occurring during intron removal Two types of phosphodiester cleavage reaction in naturally occurring ribozymes Metal ions in ribozyme catalysis Acid-base catalysis - delta ribozyme Ribosome peptidyl transferase center - entropy, substrate-assisted catalysis Crystallizing trapped reaction intermediates

Self-splicing introns
mRNA splicing in general:
Splicing always occurs in two steps: (1) Cleavage at the first exon-intron junction (2) Cleavage at the second junction and simultaneous ligation of the two exons Group I:

- Of the four known types (groups) of introns, groups I & II are selfsplicing, i.e. the splicing reaction is catalyzed by the RNA intron itself (the intron acts as a ribozyme). - The model group I intron splices itself from the pre-rRNA of tetrahymena (a ciliated protozoan). - For all group I intros, the reaction is initiated by an external nucleotide either GMP, GDP or GTP.

Naturally occurring ribozymes: Two types of rxn mechanism in RNA cleavage

- All ribozyme-mediated RNA cleavage & ligation reactions are referred to as phosphoryltransfer (or transesterification) reactions, because new phosphodiester bonds between nucleotides are made as others are simultaneously broken. General definition for non-chemists: Nucleophile = reagent that covalently bonds to a reaction partner by donating a pair of electrons (e.g. oxygen atom may be nucleophilic). Group 1 & II introns, RNAse P (left): (1) External nucleophile (guanosine) (2) 3'OH in product Small ribonucleolytic ribozymes (e.g. Hammerhead, delta, hairpin, right): (1) Internal nucleophile (2OH) (2) 2'3' cyclic intermediate (in the two mechanisms, the electrons flow in opposite direction through the phosphoryl center)

3 2

3 2

Candidate catalytic mechanisms for ribozymes

One of the intriguing aspects of ribozyme catalysis is the relative paucity of functional groups in RNA compared to protein enzymes. Potential catalytic resources could be:

(1) Bound metal ions

Some examples (dont learn by heart)

- may provide positive charge - may act as a source of water molecules - may act in acid-base catalysis *

- may act via electrostatic effects - may play direct roles in the chemistry - may act as general acids and bases * - may serve to position and/or orient the substrate(s)

(2) RNA bases of ribozyme

(3) RNA structure of ribozyme (4) Substrate assistance?

(functional groups in the substrate may be positioned by the ribozyme to catalyze the reaction) -Sounds like protein enzymes? -There is nothing new under the sun (Eclesiastes).

* Pulling protons off reactive groups, pushing them on leaving groups

(Pyle, 1993, Science 261, 709).

(1) Metal ions in ribozyme catalysis

- To orchestrate phosphoryltransfer reactions, bio-macromolecules (proteins, RNA) have harnessed the catalytic power of divalent metal ions. - Mg2+ usually coordinates six ligands in octahedral geometry: - Larger, more polarizable (soft) metals such as Mn2+ have a relatively relaxed ligand specificity, and can coordinate stably to oxygen, sulfur, nitrogen. - Smaller, less polarizable (hard) alkaline earth metals such as Mg2+ are more stringent: Mg2+ displays good affinity for oxygen, but low/nonexistent affinity for sulfur or nitrogen ligands. - RNA molecules contain a number of general ligands for the coordination of metals: Phosphate oxygens, 2'-hydroxyls, base carbonyls, providing RNA with a natural affinity for Mg2+. This affinity may explain why so many ribozymes depend on Mg2+ for structure and function (transition state oxyanions such as O- also preferentially coordinate Mg2+). - In metallo-ribozymes, the backbone of the ribozyme may serve no other function than to orientate the catalytic metal ion(s) (just as in protein metalloenzymes) - All phosphodiester bonds are intrinsically equally susceptible to attack. However, metal binding pockets or baskets in the ribozyme may direct the attack by a bound metal towards a particular phosphodiester linkage. Careful positioning of the metals may moderate the potential of ribozymes for self-destruction.

Positions in cleavage pathway where metal ions could act: Self-splicing intron

At least five positions for self-splicing intron:

(1) A metal co-ordinated hydroxyl could act as a general base, deprotonating the initial attacking nucleophile. (2) Direct metal ion coordination of a non-bridging phosphoryl oxygen may: (a) render the phosphorus center more susceptible to attack (electrophilic catalysis, left panel) (b) stabilize the substantial negative charge on the oxyanions in the transition state (center panel) (3) A co-ordinated metal may stabilize the developing negative charge on the leaving group. (4) Act as general acid, donating a proton to the leaving group. (5) Position the attacking nucleophile in the correct orientation for attacking the phosphoryl center.

Positions in cleavage pathway where metal ions could act: Hammerhead

At least four positions for hammerhead (same concepts as previous slide):

(1) A metal co-ordinated hydroxyl could act as a general base, deprotonating the 2'OH group of the initial attacking nucleophile. (2) Direct metal ion coordination of a non-bridging phosphoryl oxygen may: (a) render the phosphorus center more susceptible to attack (electrophilic catalysis, left panel) (b) stabilize the subtantial negative charge on the oxyanion in the transition state (center panel) (3) A co-ordinated metal may stabilize the developing negative charge on the leaving group. (4) Act as general acid, donating a proton to the leaving group.

Proposed general two-metal-ion mechanism for catalytic RNA

(Steitz & Steitz, 1993 PNAS 90, 6498)

Based on the observations that:

(1) Two metal ions are found at the catalytic sites of proteins involved in phosphoryl transfer reactions, spaced 4 apart (for the roles mentioned above) (2) Ribozymes require Mg2+ ions, in which co-ordination of non-bridging phosphate oxygens plays a role

It was predicted that:

(1) RNA-mediated cleavage uses the same two-metal-ion mechanism as protein-mediated RNA cleavage (2) Two metal ions would be found at ribozyme & self-splicing intron catalytic centers, spaced 4 apart (3) Since the cleavage and re-ligation steps of splicing use the same catalytic center, the same two metal ions might be used for both reactions, with the electrons flowing in opposite directions at the two steps:

More recent crystal structure for the self-splicing intron showed two metal ions in the catalytic center
SCIENCE (2005) 309, 1587.

- The crystal structure of a splicing intermediate included all metal-ion ligands and retained the ability to catalyze exon ligation (a single 2deoxy was substituted at the last nucleotide of the 5-exon to slow catalysis during crystal formation). - The location and coordination of the active-site metals were equivalent to those predicted from the generalized two metal-ion mechanism (previous slide).

- RNA enzymes and protein enzymes are not evolutionarily related, so the equivalence of group I intron and protein polymerase active sites must be convergent evolution? That macromolecular evolution arrived independently at the same solution in RNA and proteins implies an intrinsic importance of the two-metal-ion mechanism for phosphoryl transfer.

(2) General acid/base catalysis by RNA functional groups

- For general acidbase catalysis (pulling protons off reactive groups, pushing them on leaving groups), the catalytic groups should have pKa values close to pH of reaction (neutrality) to donate/accept protons readily. The pKa values of A and C are 3.5 and 4.2, respectively, and for G and U are 9.2. Therefore, these pKa values may be perturbed significantly for the bases to participate in catalysis @ physiological pH (7.5).

The hepatitis delta virus ribozyme - catalysis by an RNA base

- Crystal structures of the HDV ribozyme showed cytosine 75 adjacent to the scissile phosphate of the substrate. This suggested a role for the cytosine base in catalysis. Metal ions also required for catalysis. pH profile suggested general acid/base reaction. Two proposed mechanisms, based on two conformations seen in two different crystal structures:

- Which is correct? - Mg(H2O)6 has a dissociable proton at one of the six waters, forming Mg(H2O)5(OH-). This can act as a general base. Mg(NH2)6, however, cannot form a general base. Mg(H2O)6 is an important cofactor for reaction, whereas Mg(NH2)6 is an inhibitor. This favored upper mechanism (metal as general base) - Reaction can occur very weakly in the absence of metal, where cleavage rate now decreases with increasing pH (= general acid catalysis in absence of metal, ie. proton donation). This pH profile was particularly sensitive to mutation of C75, suggesting that C75 is the general acid. This also favored upper mechanism.

(3) Peptidyl transferase center (PTC) of the ribosome

<-<- The ribosome PTC catalyzes formation of a peptide bond (1,2) The nucleophilic -amino group of an aminoacyl-tRNA bound to the A site of the ribosome attacks the carbonyl carbon linking the peptide moiety to the P-site tRNA.

(3, 4) The resulting tetrahedral carbon intermediate decomposes to yield deacylated tRNA in the P-site and peptidyltRNA that is elongated by a single amino acid in the A site.

Transition state

Peptide-bond formation in the ribosome is catalyzed by RNA !

- This is known because there is no protein within 20 angstroms of the catalytic center (PTC) in the ribosome crystal structure. ->->->

Red - catalytic center

<-<- There are four RNA bases within hydrogen bonding distance of the catalytic center. That is the only takehome message of this figure - Mutants in these four bases showed no deficiency at all in the apparent rate constant for peptidyl transfer, suggesting that none of the rRNA bases at the PTC acts in chemical catalysis of peptide bond formation ! Yikes.

E. coli base numbering. Phosphate-yellow, O-red N-blue

Role of substrate positioning in the catalytic power of the ribosome

For the ribosome to act as a chemical catalyst, then the rate enhancement might be expected to arise from a decrease in the enthalpy of activation. If, on the other hand, the ribosome served mainly to position the substrates in the catalytic center during the PT reaction, then the rate enhancement might be expected to be largely entropic in origin. Comparison of the temperature dependence of peptide bond formation by the ribosome with that for a related uncatalyzed reaction showed that the difference was not enthalpic, but lay in a favorable entropy of activation for the ribosome-catalyzed reaction a difference of almost 60 kJ mol-1. The ratio of the two rates indicated an effective substrate concentration at the catalytic center of 105 M. Thus, the 107-fold rate enhancement produced by the ribosome is achieved in large part by a major lowering of the entropy of activation. These results strongly supported the view that the ribosome enhances the rate of peptide bond formation mainly by positioning the substrates within the active site (orientation and proximity of the substrates, exclusion of water), rather than by chemical catalysis.

Substrate-assisted catalysis?
Peptide bond formation was reduced at least 106-fold by replacement of the 2-OH of the 3 -terminal adenosine (A76) of the P site-bound tRNA with 2-H or 2-F. This suggests substrate-assisted catalysis. The 2-OH of A76 may have a role in orienting the nucleophile, stabilizing the transition state or inducing a favorable catalytic conformation of the PTC.