The hammerhead ribozyme: structure, catalysis, and gene regulation

Abstract

The hammerhead ribozyme has long been considered a prototype for understanding RNA catalysis, but discrepancies between the earlier crystal structures of a minimal hammerhead self-cleaving motif and various biochemical investigations frustrated attempt to understand hammerhead ribozyme catalysis in terms of structure. With the discovery that a tertiary contact distal from the ribozyme's active site greatly enhances its catalytic prowess, and the emergence of new corresponding crystal structures of full-length hammerhead ribozymes, a unified understanding of catalysis in terms of the structure is now possible. A mechanism in which the invariant residue G12 functions as a general base, and the 2'-OH moiety of the invariant G8, itself forming a tertiary base pair with the invariant C3, is the general acid, appears consistent with both the crystal structure and biochemical experimental results. Originally discovered in the context of plant satellite RNA viruses, the hammerhead more recently has been found embedded in the 3'-untranslated region of mature mammalian mRNAs, suggesting additional biological roles in genetic regulation.

Keywords: Catalysis; Gene regulation; Hammerhead ribozyme; RNA; Riboswitch.

Figure 1.1

The minimal and full-length hammerhead ribozymes. (A) A schematic representation of the secondary structure of the minimal hammerhead ribozyme. (B) The crystal structure of a corresponding minimal hammerhead ribozyme. The longer strand is the enzyme and the shorter strand is the substrate. (C) A schematic representation of the full-length hammerhead ribozyme emphasizing the presence of a tertiary contact between stem(s) I and II. (D) The crystal structure of a corresponding full-length hammerhead ribozyme. Again, the longer strand is the enzyme and the shorter strand is the substrate.

Figure 1.2

The active site of the full-length hammerhead ribozyme permits a mechanism to be proposed. (A) Closeup of the crystal structure of the full-length hammerhead ribozyme showing G12 positioned for general base catalysis, the 2′-OH of G8 poised for acid catalysis, and the attacking nucleophile, the 2′-O of C17, positioned for an in-line attack upon the adjacent scissile phosphate of C1.1. A9 helps to position G12 and may also engage in transition-state stabilization of the pentacoordinate oxyphosphorane transition state. (B) A mechanistic diagram illustrating partial proton dissociation and transfer in a putative transition state.

Figure 1.3

Sequence arrangement and secondary structure model of rodent CLEC2d-associated hammerhead ribozymes. Secondary structure of the mouse ribozyme sequence is shown. The rat ribozyme single nucleotide- and base pair-differences are indicated in boxes adjacent to the mouse sequence. The stop codon is denoted in white. The “substrate” sequence is shown on a gray background. The insertion sequence separating the two ribozyme segments is abridged with a thick arrow, and helices are identified by roman numerals. Rat insertion length and distance to polyA site are in italics. The predicted cleavage site is 3′ to the active site cytosine (circled).

Figure 1.4

Comparison of the CLEC2 hammerhead ribozyme sequences. Alignments of verified and predicted CLEC2 hammerhead ribozyme sequences. The sequences of the substrate and enzyme segments were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The remainders of 3′-UTRs are denoted as length of sequence in parentheses to the predicted stop codon and polyA signal. For reference, the active site cytosine is indicated with an arrowhead, and other conserved catalytic core nucleotides are boxed. Residues predicted to form base pairs in the double helices (including GU pairs) are highlighted in gray and correspond to stems indicated in the labels below the alignment. Asterisks mark nucleotides that are identical in all sequences.

Figure 1.5

MappingofCLEC2ribozymeinvariantnucleotidesonthetertiarystructureof the full-length hammerhead ribozyme. (A) Secondary structure of mCLEC2d hammer-head ribozyme. Positions conserved in all CLEC2 ribozyme sequences are circled. The cleavage site is indicated with a white arrow. (B) Positions analogous to invariant CLEC2 ribozyme nucleotides are drawn in black on the string representation of the Schistosome hammerhead ribozyme tertiary structure (PDB ID: 3ZP8). The substrate strand is represented as a wide ribbon and the site of bond cleavage is indicated with a white arrow.