my project on RIBOZYMES

RIBOZYMES__________________________________________
The structural molecular biology of ribozymes took another great leap forward during the past two years. Before ribozymes were discovered in the early 1980s, all enzymes were thought to be proteins. No detailed structural information on ribozymes became available until 1994. Now, within the past two years, near atomic resolution crystal structures are available for almost all of the known ribozymes. The latest additions include ribonuclease P, group I intron structures, the ribosome (the peptidyl transferase appears to be a ribozyme) and several smaller ribozymes, including a Diels–Alderase, the glmS ribozyme and a new hammerhead ribozyme structure that reconciles 12 years of discord. Although not all ribozymes are metalloenzymes, acid-base catalysis appears to be a universal property shared by all ribozymes as well as many of their protein cousins
Introduction
Ribozymes are enzymes whose catalytic centers are composed entirely of RNA and therefore do not require proteins for catalysis (although many exist naturally as RNA–protein complexes).
All ribozymes were believed originally to be metalloenzymes, requiringMg2+ or other divalentmetal ions for both folding and catalysis. A ‘two-metal mechanism’ had been proposed in which hydratedMg2+ ions played the roles of general acids and bases. This prediction appears to have been correct for the group I intron. Acid-base catalysis appears to be a catalytic strategy so fundamental that it occurs in both protein and RNA enzymes; in many cases, it seems that the RNA itself, rather than acting as a passive scaffold for metal ion binding, is an active participant in acid-base catalysis in the sense that nucleotide functional groups, rather than metal complexes, often mimic the roles that amino acids play in the active sites of protein enzymes. Several of the small self-cleaving RNAs as a consequence do not strictly require divalent metal ions for catalysis and no divalent metal ions have yet been observed in the
active site of the peptidyltransferase, the ribozyme that is embedded in the ribosome.


Ribonuclease P
Ribonuclease P (RNase P) was the first true RNA enzyme identified. An RNA–protein complex, the catalytic subunit of bacterial RNase P is composed entirely of RNA (and it is thought that this is the case with the eukaryotic version as well). It processes precursor tRNAs and other RNAs required for cellular metabolism.

Group I intron
The folds of the various group I introns are quite similar, permitting comparisons between molecular species. The first Azoarcus structure was in a pre-catalytic state, in which both exons (the substrate of the reaction in which adjacent exons are spliced as the intron excises itself) were present. The Tetrahymena group I intron structure represents a state in which the 30 -terminal v-guanosine and a metal ion are present in the active site. The newer structures complement these two states with an enzyme– product complex, and a complex in which all substrate, ribozyme functional groups and predicted metal ions are present in the active site.

A Diels–Alderase ribozyme
The protein Diels–Alderase is a catalytic antibody whose structure is known. The structure of a Diels–Alder ribozyme is in both the unbound and enzyme–product complex states, revealing that the ribozyme uses a combination of proximity, spatial complementarity and electronic effects to activate stereoselective catalysis, reminiscent of the protein Diels–Alderase.

The glmS ribozyme
The glmS ribozyme is a recently discovered ribozyme that is unique in the world of naturally occurring ribozymes in two respects. First, it is a ribozyme that is also a riboswitch. Second, the regulatory effector of the ribozyme, glucosamine-6-phosphate (GlcN6P), is actually a functional group that binds to the ribozyme active site and participates in the acid-base catalysis of RNA self-cleavage. The glmS ribozyme is derived from a self-cleaving RNA sequence found in the 50 -untranslatedregion (50 - UTR) of the glmS message; it cleaves itself, inactivating the message, when the cofactor GlcN6P binds. GlcN6P production is thus regulated in many Gram-positive bacteria via this ribozyme-mediated negative-feedback mechanism.

The hammerhead ribozyme
Because it is small and has a simple cleavage mechanism, the hammerhead ribozyme is perhaps the best experimentally characterized RNA enzyme, and therefore, is a clear candidate for
Structural studies. The hammerhead motif consists of three base-paired stems flanking a central core of 15 conserved nucleotides. The conserved central bases are essential for ribozyme activity. Most of these conserved bases cannot form conventional Watson-Crick base pairs, but instead form more complex structures, which mediate RNA folding and catalysis. Substitution of any of the conserved bases with other naturally occurring bases, or sometimes even artificial alteration of their functional groups, results in diminished catalytic activity. In addition, two sets of base pairs in stem III and one pair in stem II are conserved; changing these to other base pairs either impairs or abolishes catalytic function.