Molecular recognition is central to biological processes, function, and specificity. Proteins associate with ligands with a wide range of association rate constants, with maximal values matching the theoretical limit set by the rate of diffusional collision. As less is known about RNA association, we compiled association rate constants for all RNA/ligand complexes that we could find in the literature. Like proteins, RNAs exhibit a wide range of association rate constants. However, the fastest RNA association rates are considerably slower than those of the fastest protein associations and fall well below the diffusional limit. The apparently general observation of slow association with RNAs has implications for evolution and for modern-day biology. Our compilation highlights a quantitative molecular property that can contribute to biological understanding and underscores our need to develop a deeper physical understanding of molecular recognition events.

The ribosome catalyzes two fundamental biological reactions: peptidyl transfer, the formation of a peptide bond during protein synthesis, and peptidyl hydrolysis, the release of the complete protein from the peptidyl tRNA upon completion of translation. The ribosome is able to utilize and distinguish the two different nucleophiles for each reaction, the α-amine of the incoming aminoacyl tRNA versus the water molecule. The correct binding of substrates induces structural rearrangements of ribosomal active-site residues and the substrates themselves, resulting in an orientation suitable for catalysis. In addition, active-site residues appear to provide further assistance by ordering active-site water molecules and providing an electrostatic environment via a hydrogen network that stabilizes the reaction intermediates and possibly shuttles protons. Major questions remain concerning the timing, components, and mechanism of the proton transfer steps. This review summarizes the recent progress in structural, biochemical, and computational advances and presents the current mechanistic models for these two reactions.

Protein enzymes appear to use extensive packing and hydrogen-bonding interactions to precisely position catalytic groups within active sites. Due to their inherent backbone flexibility and limited side chain repertoire, RNA enzymes face additional challenges relative to proteins in precisely positioning substrates and catalytic groups. Here, we use the group I ribozyme to probe the existence, establishment, and functional consequences of an extended network of interactions in an RNA active site. The group I ribozyme catalyzes a site-specific attack of guanosine on an oligonucleotide substrate. We previously determined that the hydrogen bond between the exocyclic amino group of guanosine and the 2′-hydroxyl group at position A261 of the Tetrahymena group I ribozyme contributes to overall catalysis. We now use functional data, aided by double-mutant cycles, to probe this hydrogen bond in the individual reaction steps of the catalytic cycle. Our results indicate that this hydrogen bond is not formed upon guanosine binding to the ribozyme but instead forms at a later stage of the catalytic cycle. Formation of this hydrogen bond is correlated to other structural rearrangements in the ribozyme's active site that are promoted by docking of the oligonucleotide substrate into the ribozyme's active site, and disruption of this interaction has deleterious consequences for the chemical transformation within the ternary complex. These results, combined with earlier results, provide insight into the nature of the multiple conformational steps used by the Tetrahymena group I ribozyme to achieve its active structure and reveal an intricate, extended network of interactions that is used to establish catalytic interactions within this RNA's active site.Enzymatic reactions use the same chemical groups and functionalities that can be found on simple molecule catalysts, but the enzymatic reaction take place in specialized pockets, referred to as active sites. The protein structure provides networks of packing and hydrogen bonding interactions to precisely position substrates and catalytic groups within the active site (1). The identification and functional characterization of these networks of interactions † This work was supported by a grant from the NIH (GM 49243) to D.H. and by a grant from the Howard Hughes Medical Institute to J.A.P. *Address correspondence to this author at the Department of Biochemistry, Beckman Center, B400, Stanford University, Stanford,. herschla@stanford.edu. ⊥ Present address: Department of Biochemistry, Stanford University. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 30. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript is a fundamental step in understanding of how enzymes provide their extraordinary rate acceleration and specificity.Structural techniques, such as X-ray crystallography, are powerful tools to garner information about networks of interactions but alone are not sufficient to determine the role of specific ...

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2 ′ -and 3 ′ -deoxy (-H) and −amino (-NH 2 ) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3 ′ -OH making a nonproductive interaction with an active site metal ion termed M A and with the adjacent 2 ′ -OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M C , to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.

The determination of how enzymes achieve their catalytic power requires an understanding of how structural motifs are used to position functional groups of enzymes and substrates within active sites. The recent explosion of RNA crystal structures provides an extraordinary opportunity to delve deeply into the relationship between ribozyme structure and function. The Tetrahymena group I ribozyme provides an attractive system for such studies because of the wealth of structural information, with ten crystal structures of group I introns solved in the past