The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural, phylogenetic, and biochemical data. However, many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.

The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural, phylogenetic, and biochemical data. However, many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.

RNA molecules fold into specific three-dimensional shapes to perform structural and catalytic functions. Large RNAs can form compact globular structures, but the chemical basis for close helical packing within these molecules has been unclear. Analysis of transfer, catalysis, in vitro-selected and ribosomal RNAs reveal that helical packing predominantly involves the interaction of single-stranded adenosines with a helix minor groove. Using the Tetrahymena thermophila group I ribozyme, we show here that the near-perfect shape complementarity between the adenine base and the minor groove allows for optimal van der Waals contacts, extensive hydrogen bonding and hydrophobic surface burial, creating a highly energetically favorable interaction. Adenosine is recognized in a chemically similar fashion by a combination of protein and RNA components in the ribonucleoprotein core of the signal recognition particle. These results provide a thermodynamic explanation for the noted abundance of conserved adenosines within the unpaired regions of RNA secondary structures.

Binding of cAMP receptor protein (CRP) and CytR mediates both positive and negative control of transcription from Escherichia coli deoP2. Transcription is activated by CRP and repressed by a multi-protein CRP⅐CytR⅐CRP complex. The latter is stabilized by cooperative interactions between CRP and CytR. Similar interactions at the other transcriptional units of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. To understand the combinatorial control mechanism at deoP2, we have used quantitative footprint and gel shift analysis of CRP and CytR binding to evaluate the distribution of ligation states. By comparison to distributions for other CytRregulated promoters, we hope to understand the roles of individual states in differential gene expression. The results indicate that CytR binds specifically to multiple sites at deoP2, including both the well recognized CytR site flanked by CRP1 and CRP2 and also sites coincident with CRP1 and CRP2. Binding to these multiple sites yields both cooperative and competitive interactions between CytR and CRP. Based on these findings we propose that CytR functions as a differential modulator of CRP1 versus CRP2-mediated activation. Additional high affinity specific sites are located at deoP1 and near the middle of the 600-base pair sequence separating P1 and P2. Evaluation of the DNA sequence requirement for specific CytR binding suggests that a limited array of contiguous and overlapping CytR sites exists at deoP2. Similar extended arrays, but with different arrangements of overlapping CytR and CRP sites, are found at the other CytR-regulated promoters. We propose that competition and cooperativity in CytR and CRP binding are important to differential regulation of these promoters.In Escherichia coli, the enzymes and transport proteins required for nucleoside catabolism and recycling are encoded by genes belonging to the CytR regulon. This gene family consists of at least nine unlinked transcriptional units (for review, see Ref. 1). Expression of these transcriptional units is coordinately regulated by the interplay of two transcriptional regulatory proteins, CRP 1 (also referred to as CAP) and the CytR repressor. Transcription is activated in response to intracellular cAMP levels by CRP, repressed by CytR, and induced by cytidine. A few of the transcriptional units are also separately regulated by a second repressor, DeoR (2-4), via an independent mechanism.A key feature of the CytR regulon is that the individual cistrons are differentially expressed. Extents of activation, repression, and induction all vary among the different transcription units (cf. Ref. 5). This is achieved by nesting levels of local repression, mediated by DeoR and CytR, on a more global regulation mediated by CRP. This illustrates a process, common to both E. coli and higher order eukaryotes, in ...

Group I intron RNAs contain a core of highly conserved helices flanked by peripheral domains that stabilize the core structure. In the Tetrahymena group I ribozyme, the P4, P5, and P6 helices of the core pack tightly against a three-helix subdomain called P5abc. Chemical footprinting and the crystal structure of the Tetrahymena intron P4-P6 domain revealed that tertiary interactions between these two parts of the domain create an extensive solvent-inaccessible interface. We have examined the formation and stability of this tertiary interface by providing the P5abc segment in trans to a Tetrahymena ribozyme construct that lacks P5abc (E ∆P5abc ). Equilibrium gel shift experiments show that the affinity of the P5abc and E ∆P5abc RNAs is exceptionally strong, with a K d of ∼100 pM at 10 mM MgCl 2 (at 37°C). Chemical and enzymatic footprinting shows that the RNAs are substantially folded prior to assembly of the complex. Solvent accessibility mapping reveals that, in the absence of P5abc, the intron RNA maintains a nativelike fold but its active-site helices are not tightly packed. Upon binding of P5abc, the catalytic core becomes more tightly packed through indirect effects of the tertiary interface formation. This two-component system facilitates quantitative examination of individual tertiary contacts that stabilize the folded intron.Large RNA molecules, like proteins, readily form specific molecular shapes adapted for ligand binding and catalysis. Catalytic RNAs including group I and group II introns and RNase P have compact interiors, involving close association of several segments of secondary structure. Close packing of RNA helices requires extensive screening of the phosphodiester backbone by cations in solution (1, 2). Furthermore, noncanonical base pairs and hairpin loops provide distinct arrays of hydrogen bond donors and acceptors and irregular surface features that serve as recognition sites for RNA helices in the formation of higher order structures (3)(4)(5)(6)(7)(8). How tertiary contacts, charge screening, and other factors stabilize RNA helix packing is not well understood.The X-ray crystal structure of the P4-P6 domain from the Tetrahymena thermophila group I intron provided the first detailed view of an extended tertiary interface in RNA (9). In the 160 nucleotide domain, a flexible internal loop, J5/5a, allows the backbone to make a ∼180°bend, enabling parallel association of two coaxially stacked helical segments (Figure 1). One-half of the P4-P6 domain, a three-helix subdomain called P5abc, binds at two distinct locations to the other half of the domain, constituted by the coaxially stacked P5, P4, and P6 helices (Figure 1). The A-rich bulge of P5abc contacts the minor groove of helix P4 by backbone and base-mediated hydrogen bonds. Additionally, a GAAA tetraloop in P5abc docks into its tetraloop receptor by both cross-helical base stacking and extensive hydrogen bonding. Similar GAAA tetraloop/tetraloop receptor interactions are thought to occur in other large RNAs including group I...