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Approaches To Understanding Catalysis by RNA
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:
Self-splicing:
- 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.
3 2
3 2
- 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)
(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).
- 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
(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
(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.
- 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, 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
How?
<-<- 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.
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.